Heteroaryl pyridone and aza-pyridone compounds with electrophilic functionality

ABSTRACT

Heteroaryl pyridone and aza-pyridone amide compounds with electrophilic functionality of Formula I are provided, including stereoisomers, tautomers, and pharmaceutically acceptable salts thereof, useful for inhibiting Btk, and for treating cancer and immune disorders such as inflammation mediated by Btk. Methods of using compounds of Formula I for in vitro, in situ, and in vivo diagnosis, and treatment of such disorders in mammalian cells, or associated pathological conditions, are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This non-provisional application filed under 37 CFR §1.53(b), claims thebenefit under 35 USC §119(e) of U.S. Provisional Application Ser. No.61/912,147 filed on 5 Dec. 2013, which is incorporated by reference inentirety.

FIELD OF THE INVENTION

The invention relates generally to compounds for treating disordersmediated by Bruton's Tyrosine Kinase (Btk) including inflammation,immunological, and cancer, and more specifically to compounds whichinhibit Btk activity. The invention also relates to methods of using thecompounds for in vitro, in situ, and in vivo diagnosis or treatment ofmammalian cells, or associated pathological conditions.

BACKGROUND OF THE INVENTION

Protein kinases, the largest family of human enzymes, encompass wellover 500 proteins. Bruton's Tyrosine Kinase (Btk) is a member of the Tecfamily of tyrosine kinases, and is a regulator of early B-celldevelopment as well as mature B-cell activation, signaling, andsurvival.

B-cell signaling through the B-cell receptor (BCR) can lead to a widerange of biological outputs, which in turn depend on the developmentalstage of the B-cell. The magnitude and duration of BCR signals must beprecisely regulated. Aberrant BCR-mediated signaling can causedisregulated B-cell activation and/or the formation of pathogenicauto-antibodies leading to multiple autoimmune and/or inflammatorydiseases. Mutation of Btk in humans results in X-linkedagammaglobulinaemia (XLA). This disease is associated with the impairedmaturation of B-cells, diminished immunoglobulin production, compromisedT-cell-independent immune responses and marked attenuation of thesustained calcium sign upon BCR stimulation. Evidence for the role ofBtk in allergic disorders and/or autoimmune disease and/or inflammatorydisease has been established in Btk-deficient mouse models. For example,in standard murine preclinical models of systemic lupus erythematosus(SLE), Btk deficiency has been shown to result in a marked ameliorationof disease progression. Moreover, Btk deficient mice can also beresistant to developing collagen-induced arthritis and can be lesssusceptible to Staphylococcus-induced arthritis. A large body ofevidence supports the role of B-cells and the humoral immune system inthe pathogenesis of autoimmune and/or inflammatory diseases.Protein-based therapeutics (such as Rituxan®, Genentech/Biogen Idec)developed to deplete B-cells, represent an approach to the treatment ofa number of autoimmune and/or inflammatory diseases. Because of Btk'srole in B-cell activation, inhibitors of Btk can be useful as inhibitorsof B-cell mediated pathogenic activity (such as autoantibodyproduction). Btk is also expressed in osteoclasts, mast cells andmonocytes and has been shown to be important for the function of thesecells. For example, Btk deficiency in mice is associated with impairedIgE-mediated mast cell activation (marked diminution of TNF-alpha andother inflammatory cytokine release), and Btk deficiency in humans isassociated with greatly reduced TNF-alpha production by activatedmonocytes.

Thus, inhibition of Btk activity can be useful for the treatment ofallergic disorders and/or autoimmune and/or inflammatory diseases suchas: SLE, rheumatoid arthritis, multiple vasculitides, idiopathicthrombocytopenic purpura (ITP), myasthenia gravis, allergic rhinitis,and asthma (Di Paolo et al (2011) Nature Chem. Biol. 7(1):41-50; Liu(2011) Drug Metab. and Disposition 39(10):1840-1849; Liu et al (2011)Jour. of Pharm. and Exper. Ther. 338(1):154-163; Lou et al (2012) J.Med. Chem. 55(10):4539-4550; Farooqui et al (2013) Expert Opinion onOrphan Drugs, Volume: 1, Issue: 11: 925-933). In addition, Btk has beenreported to play a role in apoptosis; thus, inhibition of Btk activitycan be useful for cancer, as well as the treatment of B-cell lymphoma,leukemia, and other hematological malignancies (U.S. Pat. No.7,514,444). Given the role of Btk in osteoclast function, the inhibitionof Btk activity can be useful for the treatment of bone disorders suchas osteoporosis. Specific Btk inhibitors have been reported (U.S. Pat.No. 7,884,108, WO 2010/056875; U.S. Pat. No. 7,405,295; U.S. Pat. No.7,393,848; WO 2006/053121; U.S. Pat. No. 7,947,835; US 2008/0139557;U.S. Pat. No. 7,838,523; US 2012/0040949; US 2012/0295885; US2013/0045965; U.S. Pat. No. 7,683,064; U.S. Pat. No. 7,902,194; U.S.Pat. No. 7,906,509; U.S. Pat. No. 8,124,604; US 2008/0125417; US2011/0118233; WO2011/140488; US 2012/0010191; WO2013/067274; US2013/0116235; WO2013/067277; US 2013/0116245; WO2013/067260; US2013/0116262; WO2013/067264; US 2013/0116246.

Irreversible inhibitors provide potent and selective inhibition oftyrosine kinase enzymes and may overcome the tumor resistanceencountered with reversible tyrosine kinase inhibitors (Carmi et al(2012) Biochem. Pharmacol. 84(11):1388-1399). The perceived advantagesof irreversible target-binding by covalent bond formation of inhibitorwith target are efficacy, ability to overcome competition, andwithin-class selectivity. Intrinsic liabilities include target- andmutation-dependent responses and toxicity. Irreversible inhibitorsinactivate their protein target through covalent interaction with anucleophilic cysteine residue within the nucleotide binding pocket ofthe kinase domain. Different irreversible tyrosine kinase inhibitorsdirected against epidermal growth factor receptor (EGFR), Bruton'styrosine kinase (Btk), vascular endothelial growth factor receptor(VEGFR) and fibroblast growth factor receptor tyrosine kinase (FGFR)have been developed and some of them have been employed clinically asanticancer agents

Compounds that form covalent bonds with Btk have been reported (U.S.Pat. No. 7,514,444; U.S. Pat. No. 8,088,781), including ibrutinib(IMBRUVICA®, Pharmacyclics, Sunnyvale, Calif., Janssen Biotech, Inc.,Raritan, N.J.) which has been approved by the FDA to treat patients withB-cell malignancy, mantle cell lymphoma (MCL). Ibrutinib has alsodemonstrated clinical efficacy in chronic lymphocytic leukemia (CLL) andsmall lymphocytic lymphoma (SLL). Also, PF-112 (Pfizer, Inc.) is acovalent-reversible inhibitor of Btk being developed for the treatmentof autoimmune and inflammatory disease.

SUMMARY OF THE INVENTION

The invention relates generally to heteroaryl pyridone and aza-pyridoneamide compounds with electrophilic functionality and Bruton's TyrosineKinase (Btk) modulating activity having the Formula I structure:

including stereoisomers, tautomers, or pharmaceutically acceptable saltsthereof. The various substituents are defined herein.

One aspect of the invention is a Formula I compound that bindscovalently to Btk.

Another aspect of the invention is a Formula I compound selective forbinding to Btk and tyrosine kinases having a cysteine residue in anamino acid sequence position of the tyrosine kinase that is homologousto the amino acid sequence position of cysteine 481 in Btk.

In one embodiment, a Formula I compound selectively and irreversiblyinhibits an activated form of its target tyrosine kinase (e.g., aphosphorylated form of the tyrosine kinase). For example, activated Btkis transphosphorylated at tyrosine 551. Thus, in these embodiments theirreversible Btk inhibitor inhibits the target kinase in cells only oncethe target kinase is activated by the signaling events.

In an exemplary embodiment, the Formula I compound has a Michaelacceptor moiety.

Another aspect of the invention is a pharmaceutical compositioncomprised of a Formula I compound and a pharmaceutically acceptablecarrier, glidant, diluent, or excipient. The pharmaceutical compositionmay further comprise a second therapeutic agent.

Another aspect of the invention is a process for making a pharmaceuticalcomposition which comprises combining a Formula I compound with apharmaceutically acceptable carrier, glidant, diluent, or excipient.

Another aspect of the invention is a method of treating a disease ordisorder which method comprises administering a therapeuticallyeffective amount of a Formula I compound to a patient with a disease ordisorder selected from immune disorders, cancer, cardiovascular disease,viral infection, inflammation, metabolism/endocrine function disordersand neurological disorders, and mediated by Bruton's tyrosine kinase.

The invention includes a kit for treating a condition mediated byBruton's tyrosine kinase, comprising: a) a first pharmaceuticalcomposition comprising a Formula I compound; and b) instructions foruse.

The invention includes a Formula I compound for use in combination withan additional therapeutic agent in treating a disease or disorder.

The invention includes methods of making a Formula I compound.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingstructures and formulas. While the invention will be described inconjunction with the enumerated embodiments, it will be understood thatthey are not intended to limit the invention to those embodiments. Onthe contrary, the invention is intended to cover all alternatives,modifications, and equivalents which may be included within the scope ofthe present invention as defined by the claims. One skilled in the artwill recognize many methods and materials similar or equivalent to thosedescribed herein, which could be used in the practice of the presentinvention. The present invention is in no way limited to the methods andmaterials described. In the event that one or more of the incorporatedliterature, patents, and similar materials differs from or contradictsthis application, including but not limited to defined terms, termusage, described techniques, or the like, this application controls.Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. The nomenclature used in this Application is based on IUPACsystematic nomenclature, unless indicated otherwise.

DEFINITIONS

Definition of standard chemistry terms may be found in reference works,including McMurry “ORGANIC CHEMISTRY Fifth ED.” (2000) Brooks/Cole,Pacific Grove.

When indicating the number of substituents, the term “one or more”refers to the range from one substituent to the highest possible numberof substitution, i.e. replacement of one hydrogen up to replacement ofall hydrogen atoms by substituents. The term “substituent” denotes anatom or a group of atoms replacing a hydrogen atom on the parentmolecule. The term “substituted” denotes that a specified group bearsone or more substituents. Where any group may carry multiplesubstituents and a variety of possible substituents is provided, thesubstituents are independently selected and need not to be the same. Theterm “unsubstituted” means that the specified group bears nosubstituents. The term “optionally substituted” means that the specifiedgroup is unsubstituted or substituted by one or more substituents,independently chosen from the group of possible substituents. Whenindicating the number of substituents, the term “one or more” means fromone substituent to the highest possible number of substitution, i.e.replacement of one hydrogen atom up to replacement of all hydrogen atomsby substituents.

The term “alkyl” as used herein refers to a saturated linear orbranched-chain monovalent hydrocarbon radical of one to twelve carbonatoms (C₁-C₁₂), wherein the alkyl radical may be optionally substitutedindependently with one or more substituents described below. In anotherembodiment, an alkyl radical is one to eight carbon atoms (C₁-C₈), orone to six carbon atoms (C₁-C₆). Examples of alkyl groups include, butare not limited to, methyl (Me, —CH₃), ethyl (Et, —CH₂CH₃), 1-propyl(n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr, i-propyl, —CH(CH₃)₂),1-butyl (n-Bu, n-butyl, —CH₂CH₂CH₂CH₃), 2-methyl-1-propyl (i-Bu,i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl, —CH(CH₃)CH₂CH₃),2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl (n-pentyl,—CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃, 1-heptyl, 1-octyl, and the like.

The term “alkylene” as used herein refers to a saturated linear orbranched-chain divalent hydrocarbon radical of one to twelve carbonatoms (C₁-C₁₂), wherein the alkylene radical may be optionallysubstituted independently with one or more substituents described below.In another embodiment, an alkylene radical is one to eight carbon atoms(C₁-C₈), or one to six carbon atoms (C₁-C₆). Examples of alkylene groupsinclude, but are not limited to, methylene (—CH₂—), ethylene (—CH₂CH₂—),propylene (—CH₂CH₂CH₂—), and the like.

The term “alkenyl” refers to linear or branched-chain monovalenthydrocarbon radical of two to eight carbon atoms (C₂-C₈) with at leastone site of unsaturation, i.e., a carbon-carbon, sp² double bond,wherein the alkenyl radical may be optionally substituted independentlywith one or more substituents described herein, and includes radicalshaving “cis” and “trans” orientations, or alternatively, “E” and “Z”orientations. Examples include, but are not limited to, ethylenyl orvinyl (—CH═CH₂), allyl (—CH₂CH═CH₂), and the like.

The term “alkenylene” refers to linear or branched-chain divalenthydrocarbon radical of two to eight carbon atoms (C₂-C₈) with at leastone site of unsaturation, i.e., a carbon-carbon, sp² double bond,wherein the alkenylene radical may be optionally substitutedindependently with one or more substituents described herein, andincludes radicals having “cis” and “trans” orientations, oralternatively, “E” and “Z” orientations. Examples include, but are notlimited to, ethylenylene or vinylene (—CH═CH—), allyl (—CH₂CH═CH—), andthe like.

The term “alkynyl” refers to a linear or branched monovalent hydrocarbonradical of two to eight carbon atoms (C₂-C₈) with at least one site ofunsaturation, i.e., a carbon-carbon, sp triple bond, wherein the alkynylradical may be optionally substituted independently with one or moresubstituents described herein. Examples include, but are not limited to,ethynyl (—C≡CH), propynyl (propargyl, —CH₂C≡CH), and the like.

The term “alkynylene” refers to a linear or branched divalenthydrocarbon radical of two to eight carbon atoms (C₂-C₈) with at leastone site of unsaturation, i.e., a carbon-carbon, sp triple bond, whereinthe alkynylene radical may be optionally substituted independently withone or more substituents described herein. Examples include, but are notlimited to, ethynylene (—C≡C—), propynylene (propargylene, —CH₂C≡C—),and the like.

The terms “carbocycle”, “carbocyclyl”, “carbocyclic ring” and“cycloalkyl” refer to a monovalent non-aromatic, saturated or partiallyunsaturated ring having 3 to 12 carbon atoms (C₃-C₁₂) as a monocyclicring or 7 to 12 carbon atoms as a bicyclic ring. Bicyclic carbocycleshaving 7 to 12 atoms can be arranged, for example, as a bicyclo[4,5],[5,5], [5,6] or [6,6] system, and bicyclic carbocycles having 9 or 10ring atoms can be arranged as a bicyclo[5,6] or [6,6] system, or asbridged systems such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane andbicyclo[3.2.2]nonane. Spiro moieties are also included within the scopeof this definition. Examples of monocyclic carbocycles include, but arenot limited to, cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl,cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl,cycloundecyl, cyclododecyl, and the like. Carbocyclyl groups areoptionally substituted independently with one or more substituentsdescribed herein.

“Aryl” means a monovalent aromatic hydrocarbon radical of 6-20 carbonatoms (C₆-C₂₀) derived by the removal of one hydrogen atom from a singlecarbon atom of a parent aromatic ring system. Some aryl groups arerepresented in the exemplary structures as “Ar”. Aryl includes bicyclicradicals comprising an aromatic ring fused to a saturated, partiallyunsaturated ring, or aromatic carbocyclic ring. Typical aryl groupsinclude, but are not limited to, radicals derived from benzene (phenyl),substituted benzenes, naphthalene, anthracene, biphenyl, indenyl,indanyl, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, and thelike. Aryl groups are optionally substituted independently with one ormore substituents described herein.

“Arylene” means a divalent aromatic hydrocarbon radical of 6-20 carbonatoms (C₆-C₂₀) derived by the removal of two hydrogen atom from a twocarbon atoms of a parent aromatic ring system. Some arylene groups arerepresented in the exemplary structures as “Ar”. Arylene includesbicyclic radicals comprising an aromatic ring fused to a saturated,partially unsaturated ring, or aromatic carbocyclic ring. Typicalarylene groups include, but are not limited to, radicals derived frombenzene (phenylene), substituted benzenes, naphthalene, anthracene,biphenylene, indenylene, indanylene, 1,2-dihydronaphthalene,1,2,3,4-tetrahydronaphthyl, and the like. Arylene groups are optionallysubstituted with one or more substituents described herein.

The terms “heterocycle”, “heterocyclyl” and “heterocyclic ring” are usedinterchangeably herein and refer to a saturated or a partiallyunsaturated (i.e., having one or more double and/or triple bonds withinthe ring) carbocyclic radical of 3 to about 20 ring atoms in which atleast one ring atom is a heteroatom selected from nitrogen, oxygen,phosphorus and sulfur, the remaining ring atoms being C, where one ormore ring atoms is optionally substituted independently with one or moresubstituents described below. A heterocycle may be a monocycle having 3to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selectedfrom N, O, P, and S) or a bicycle having 7 to 10 ring members (4 to 9carbon atoms and 1 to 6 heteroatoms selected from N, O, P, and S), forexample: a bicyclo[4,5], [5,5], [5,6], or [6,6] system. Heterocycles aredescribed in Paquette, Leo A.; “Principles of Modern HeterocyclicChemistry” (W. A. Benjamin, N.Y., 1968), particularly Chapters 1, 3, 4,6, 7, and 9; “The Chemistry of Heterocyclic Compounds, A series ofMonographs” (John Wiley & Sons, New York, 1950 to present), inparticular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960)82:5566. “Heterocyclyl” also includes radicals where heterocycleradicals are fused with a saturated, partially unsaturated ring, oraromatic carbocyclic or heterocyclic ring. Examples of heterocyclicrings include, but are not limited to, morpholin-4-yl, piperidin-1-yl,piperazinyl, piperazin-4-yl-2-one, piperazin-4-yl-3-one,pyrrolidin-1-yl, thiomorpholin-4-yl, S-dioxothiomorpholin-4-yl,azocan-1-yl, azetidin-1-yl, octahydropyrido[1,2-a]pyrazin-2-yl,[1,4]diazepan-1-yl, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl,tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino,thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl,thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl,thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl,4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl,dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl,pyrazolidinylimidazolinyl, imidazolidinyl, 3-azabicyco[3.1.0]hexanyl,3-azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexanyl, 3H-indolylquinolizinyl and N-pyridyl ureas. Spiro moieties are also includedwithin the scope of this definition. Examples of a heterocyclic groupwherein 2 ring atoms are substituted with oxo (═O) moieties arepyrimidinonyl and 1,1-dioxo-thiomorpholinyl. The heterocycle groupsherein are optionally substituted independently with one or moresubstituents described herein.

The term “heteroaryl” refers to a monovalent aromatic radical of 5-, 6-,or 7-membered rings, and includes fused ring systems (at least one ofwhich is aromatic) of 5-20 atoms, containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur. Examples ofheteroaryl groups are pyridinyl (including, for example,2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl(including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl,pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl,oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl,isoquinolinyl, tetrahydroisoquinolinyl, indolyl, benzimidazolyl,benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl,pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl,triazolyl, thiadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl,benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl,quinoxalinyl, naphthyridinyl, and furopyridinyl. Heteroaryl groups areoptionally substituted independently with one or more substituentsdescribed herein.

The heterocycle or heteroaryl groups may be carbon (carbon-linked), ornitrogen (nitrogen-linked) bonded where such is possible. By way ofexample and not limitation, carbon bonded heterocycles or heteroarylsare bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5,or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline.

By way of example and not limitation, nitrogen bonded heterocycles orheteroaryls are bonded at position 1 of an aziridine, azetidine,pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole,imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline,2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline,1H-indazole, position 2 of a isoindole, or isoindoline, position 4 of amorpholine, and position 9 of a carbazole, or β-carboline.

The term “Michael acceptor moiety” refers to a functional group that canparticipate in a Michael reaction, wherein a new covalent bond is formedbetween a portion of the Michael acceptor moiety and the donor moiety.The Michael acceptor moiety is an electrophile and the “donor moiety” isa nucleophile. The “Q” groups presented in Formula I compounds arenon-limiting examples of Michael acceptor moieties.

The term “nucleophile” or “nucleophilic” refers to an electron richcompound, or moiety thereof. An example of a nucleophile includes, butin no way is limited to, a cysteine residue of a molecule, such as, forexample Cys 481 of Btk.

The term “electrophile”, or “electrophilic” refers to an electron pooror electron deficient molecule, or moiety thereof. Examples ofelectrophiles include, but in no way are limited to, Michael acceptormoieties such as α,β-unsaturated acyl functional groups, such asacrylamides, acrylate esters, α,β-unsaturated ketones, acrylonitriles,and α,β-unsaturated nitros.

The term “irreversible inhibitor” means a targeted compound which formsa stable covalent bond through a reactive functional group and directlylinked to a recognition portion of the target. Irreversible inhibitorsof Btk may inactivate their Btk target through covalent interaction witha nucleophilic cysteine residue within the nucleotide binding pocket ofthe kinase domain of Btk.

The terms “treat” and “treatment” refer to therapeutic treatment,wherein the object is to slow down (lessen) an undesired physiologicalchange or disorder, such as the development or spread of arthritis orcancer. For purposes of this invention, beneficial or desired clinicalresults include, but are not limited to, alleviation of symptoms,diminishment of extent of disease, stabilized (i.e., not worsening)state of disease, delay or slowing of disease progression, ameliorationor palliation of the disease state, and remission (whether partial ortotal), whether detectable or undetectable. “Treatment” can also meanprolonging survival as compared to expected survival if not receivingtreatment. Those in need of treatment include those with the conditionor disorder.

The phrase “therapeutically effective amount” means an amount of acompound of the present invention that (i) treats the particulardisease, condition, or disorder, (ii) attenuates, ameliorates, oreliminates one or more symptoms of the particular disease, condition, ordisorder, or (iii) prevents or delays the onset of one or more symptomsof the particular disease, condition, or disorder described herein. Inthe case of cancer, the therapeutically effective amount of the drug mayreduce the number of cancer cells; reduce the tumor size; inhibit (i.e.,slow to some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. To the extent the drug may prevent growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy,efficacy can be measured, for example, by assessing the time to diseaseprogression (TTP) and/or determining the response rate (RR).

“Inflammatory disorder” as used herein can refer to any disease,disorder, or syndrome in which an excessive or unregulated inflammatoryresponse leads to excessive inflammatory symptoms, host tissue damage,or loss of tissue function. “Inflammatory disorder” also refers to apathological state mediated by influx of leukocytes and/or neutrophilchemotaxis.

“Inflammation” as used herein refers to a localized, protective responseelicited by injury or destruction of tissues, which serves to destroy,dilute, or wall off (sequester) both the injurious agent and the injuredtissue. Inflammation is notably associated with influx of leukocytesand/or neutrophil chemotaxis. Inflammation can result from infectionwith pathogenic organisms and viruses and from noninfectious means suchas trauma or reperfusion following myocardial infarction or stroke,immune response to foreign antigen, and autoimmune responses.Accordingly, inflammatory disorders amenable to treatment with Formula Icompounds encompass disorders associated with reactions of the specificdefense system as well as with reactions of the nonspecific defensesystem.

“Specific defense system” refers to the component of the immune systemthat reacts to the presence of specific antigens. Examples ofinflammation resulting from a response of the specific defense systeminclude the classical response to foreign antigens, autoimmune diseases,and delayed type hypersensitivity response mediated by T-cells. Chronicinflammatory diseases, the rejection of solid transplanted tissue andorgans, e.g., kidney and bone marrow transplants, and graft versus hostdisease (GVHD), are further examples of inflammatory reactions of thespecific defense system.

The term “nonspecific defense system” as used herein refers toinflammatory disorders that are mediated by leukocytes that areincapable of immunological memory (e.g., granulocytes, and macrophages).Examples of inflammation that result, at least in part, from a reactionof the nonspecific defense system include inflammation associated withconditions such as adult (acute) respiratory distress syndrome (ARDS) ormultiple organ injury syndromes; reperfusion injury; acuteglomerulonephritis; reactive arthritis; dermatoses with acuteinflammatory components; acute purulent meningitis or other centralnervous system inflammatory disorders such as stroke; thermal injury;inflammatory bowel disease; granulocyte transfusion associatedsyndromes; and cytokine-induced toxicity.

“Autoimmune disease” as used herein refers to any group of disorders inwhich tissue injury is associated with humoral or cell-mediatedresponses to the body's own constituents.

“Allergic disease” as used herein refers to any symptoms, tissue damage,or loss of tissue function resulting from allergy. “Arthritic disease”as used herein refers to any disease that is characterized byinflammatory lesions of the joints attributable to a variety ofetiologies. “Dermatitis” as used herein refers to any of a large familyof diseases of the skin that are characterized by inflammation of theskin attributable to a variety of etiologies. “Transplant rejection” asused herein refers to any immune reaction directed against graftedtissue, such as organs or cells (e.g., bone marrow), characterized by aloss of function of the grafted and surrounding tissues, pain, swelling,leukocytosis, and thrombocytopenia. The therapeutic methods of thepresent invention include methods for the treatment of disordersassociated with inflammatory cell activation.

“Inflammatory cell activation” refers to the induction by a stimulus(including, but not limited to, cytokines, antigens or auto-antibodies)of a proliferative cellular response, the production of solublemediators (including but not limited to cytokines, oxygen radicals,enzymes, prostanoids, or vasoactive amines), or cell surface expressionof new or increased numbers of mediators (including, but not limited to,major histocompatibility antigens or cell adhesion molecules) ininflammatory cells (including but not limited to monocytes, macrophages,T lymphocytes, B lymphocytes, granulocytes (i.e., polymorphonuclearleukocytes such as neutrophils, basophils, and eosinophils), mast cells,dendritic cells, Langerhans cells, and endothelial cells). It will beappreciated by persons skilled in the art that the activation of one ora combination of these phenotypes in these cells can contribute to theinitiation, perpetuation, or exacerbation of an inflammatory disorder.

The term “NSAID” is an acronym for “non-steroidal anti-inflammatorydrug” and is a therapeutic agent with analgesic, antipyretic (loweringan elevated body temperature and relieving pain without impairingconsciousness) and, in higher doses, with anti-inflammatory effects(reducing inflammation). The term “non-steroidal” is used to distinguishthese drugs from steroids, which (among a broad range of other effects)have a similar eicosanoid-depressing, anti-inflammatory action. Asanalgesics, NSAIDs are unusual in that they are non-narcotic. NSAIDsinclude aspirin, ibuprofen, and naproxen. NSAIDs are usually indicatedfor the treatment of acute or chronic conditions where pain andinflammation are present. NSAIDs are generally indicated for thesymptomatic relief of the following conditions: rheumatoid arthritis,osteoarthritis, inflammatory arthropathies (e.g. ankylosing spondylitis,psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhoea,metastatic bone pain, headache and migraine, postoperative pain,mild-to-moderate pain due to inflammation and tissue injury, pyrexia,ileus, and renal colic. Most NSAIDs act as non-selective inhibitors ofthe enzyme cyclooxygenase, inhibiting both the cyclooxygenase-1 (COX-1)and cyclooxygenase-2 (COX-2) isoenzymes. Cyclooxygenase catalyzes theformation of prostaglandins and thromboxane from arachidonic acid(itself derived from the cellular phospholipid bilayer by phospholipaseA₂). Prostaglandins act (among other things) as messenger molecules inthe process of inflammation. COX-2 inhibitors include celecoxib,etoricoxib, lumiracoxib, parecoxib, rofecoxib, rofecoxib, andvaldecoxib.

The terms “cancer” refers to or describe the physiological condition inmammals that is typically characterized by unregulated cell growth. A“tumor” comprises one or more cancerous cells. Examples of cancerinclude, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma,and leukemia or lymphoid malignancies. More particular examples of suchcancers include squamous cell cancer (e.g., epithelial squamous cellcancer), lung cancer including small-cell lung cancer, non-small celllung cancer (“NSCLC”), adenocarcinoma of the lung and squamous carcinomaof the lung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectalcancer, endometrial or uterine carcinoma, salivary gland carcinoma,kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head andneck cancer.

“Hematological malignancies” (British spelling “Haematological”malignancies) are the types of cancer that affect blood, bone marrow,and lymph nodes. As the three are intimately connected through theimmune system, a disease affecting one of the three will often affectthe others as well: although lymphoma is a disease of the lymph nodes,it often spreads to the bone marrow, affecting the blood. Hematologicalmalignancies are malignant neoplasms (“cancer”), and they are generallytreated by specialists in hematology and/or oncology. In some centers“Hematology/oncology” is a single subspecialty of internal medicinewhile in others they are considered separate divisions (there are alsosurgical and radiation oncologists). Not all hematological disorders aremalignant (“cancerous”); these other blood conditions may also bemanaged by a hematologist. Hematological malignancies may derive fromeither of the two major blood cell lineages: myeloid and lymphoid celllines. The myeloid cell line normally produces granulocytes,erythrocytes, thrombocytes, macrophages and mast cells; the lymphoidcell line produces B, T, NK and plasma cells. Lymphomas, lymphocyticleukemias, and myeloma are from the lymphoid line, while acute andchronic myelogenous leukemia, myelodysplastic syndromes andmyeloproliferative diseases are myeloid in origin. Leukemias includeAcute lymphoblastic leukemia (ALL), Acute myelogenous leukemia (AML),Chronic lymphocytic leukemia (CLL), Chronic myelogenous leukemia (CML),Acute monocytic leukemia (AMOL) and small lymphocytic lymphoma (SLL).Lymphomas include Hodgkin's lymphomas (all four subtypes) andNon-Hodgkin's lymphomas (NHL, all subtypes), mantle cell lymphoma,diffuse large B-cell lymphoma, and follicular lymphoma. Hematologicalmalignancies also include Waldenstrom's macroglobulinemia and multiplemyeloma.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer, regardless of mechanism of action. Classes ofchemotherapeutic agents include, but are not limited to: alkylatingagents, antimetabolites, spindle poison plant alkaloids,cytotoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies,photosensitizers, and kinase inhibitors. Chemotherapeutic agents includecompounds used in “targeted therapy” and conventional chemotherapy.Examples of chemotherapeutic agents include: ibrutinib (IMBRUVICA®,PCI-32765, Pharmacyclics Inc.; CAS Reg. No. 936563-96-1, U.S. Pat. No.7,514,444), idelalisib (formerly CAL-101, GS 1101, GS-1101, GileadSciences Inc.; CAS Reg. No. 1146702-54-6), erlotinib (TARCEVA®,Genentech/OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU(fluorouracil, 5-fluorouracil, CAS Reg. No. 51-21-8), gemcitabine(GEMZAR®, Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin(cis-diamine, dichloroplatinum(II), CAS No. 15663-27-1), carboplatin(CAS No. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology,Princeton, N.J.), trastuzumab (HERCEPTIN®, Genentech), temozolomide(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide,CAS No. 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethanamine,NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCINO), Akti-1/2,HPPD, and rapamycin.

Chemotherapeutic agents include a Bcl-2 inhibitor, a Btk inhibitor, aJAK inhibitor, a Syk inhibitor; a Tyk inhibitor, and an anti-CD20antibody.

A bcl-2 inhibitor for use in combination with a compound of theinvention is venetoclax (CAS Reg. No. 1257044-40-8; ABT-199; GDC-0199;RG-7601, AbbVie Inc., Genentech Inc.). Venetoclax is a so-calledBH3-mimetic drug designed to block the function of the protein Bcl 2 andis in Phase 3 clinical trials for the treatment of Multiple myeloma,Chronic lymphocytic leukemia, Systemic lupus erythematosus, Diffuselarge B-cell lymphoma, Acute myelogenous leukemia, and Non-Hodgkinlymphoma (Souers et al Nat Med. 2013 Jan. 6. doi: 10.1038/nm.3048).Venetoclax has the IUPAC name:4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide.

An anti-CD20 antibody for use in combination with a compound of theinvention is obinutuzumab (CAS Reg. No. 949142-50-1; GAZYVA®, GenentechInc.) approved by the US FDA in 2013, for the treatment of chroniclymphocytic leukemia in combination with chemotherapy in treatment-naivepatients. Obinutuzumab targets the B-lymphocyte antigen CD20 and treatsChronic lymphocytic leukemia, Diffuse large B-cell lymphoma, Non-Hodgkinlymphoma, Follicle center lymphoma, and Mantle cell lymphoma.

More examples of chemotherapeutic agents include: oxaliplatin(ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent(SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinibmesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, AstraZeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235(PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin(folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib(TYKERB®, GSK572016, Glaxo Smith Kline), lonafarnib (SARASAR™, SCH66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs),gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11,Pfizer), tipifarnib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™(Cremophor-free), albumin-engineered nanoparticle formulations ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, II),vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chlorambucil, AG1478,AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib(GlaxoSmithKline), canfosfamide (TELCYTA®, Telik), thiotepa andcyclosphosphamide (CYTOXAN®, NEOSAR®); alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analog topotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogs, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, calicheamicin gamma1I, calicheamicin omegaI1 (Angew Chem.Intl. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, nemorubicin,marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; 6-thioguanine;mercaptopurine; methotrexate; platinum analogs such as cisplatin andcarboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine (NAVELBINE®); novantrone; teniposide;edatrexate; daunomycin; aminopterin; capecitabine (XELODA®, Roche);ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoids such as retinoic acid; andpharmaceutically acceptable salts, acids and derivatives of any of theabove.

Also included in the definition of “chemotherapeutic agent” are: (i)anti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptor modulators(SERMs), including, for example, tamoxifen (including NOLVADEX®;tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifinecitrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase,which regulates estrogen production in the adrenal glands, such as, forexample, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrolacetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole,RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX®(anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide,nilutamide, bicalutamide, leuprolide, and goserelin; as well astroxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) proteinkinase inhibitors such as MEK inhibitors (WO 2007/044515); (v) lipidkinase inhibitors; (vi) antisense oligonucleotides, particularly thosewhich inhibit expression of genes in signaling pathways implicated inaberrant cell proliferation, for example, PKC-alpha, Raf and H-Ras, suchas oblimersen (GENASENSE®, Genta Inc.); (vii) ribozymes such as VEGFexpression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors;(viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®,LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; topoisomerase 1 inhibitorssuch as LURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such asbevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptablesalts, acids and derivatives of any of the above.

Also included in the definition of “chemotherapeutic agent” aretherapeutic antibodies such as alemtuzumab (Campath), bevacizumab(AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab(VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec),pertuzumab (OMNITARG™, 2C4, Genentech), trastuzumab (HERCEPTIN®,Genentech), tositumomab (Bexxar, Corixia), and the antibody drugconjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth).

Humanized monoclonal antibodies with therapeutic potential aschemotherapeutic agents in combination with the Btk inhibitors of theinvention include: alemtuzumab, apolizumab, aselizumab, atlizumab,bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumabmertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab,daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab,fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab,labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab,motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab,ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab,pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab,reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab,sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan,tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab,trastuzumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab,urtoxazumab, and visilizumab.

A “metabolite” is a product produced through metabolism in the body of aspecified compound or salt thereof. Metabolites of a compound may beidentified using routine techniques known in the art and theiractivities determined using tests such as those described herein. Suchproducts may result for example from the oxidation, reduction,hydrolysis, amidation, de-amidation, esterification, de-esterification,enzymatic cleavage, and the like, of the administered compound. Ametabolite may also be formed by reversal of the Michael addition of thecysteine thiol of Btk to the electrophilic functionality of the FormulaI compound. Accordingly, the invention includes metabolites of compoundsof the invention, including compounds produced by a process comprisingcontacting a Formula I compound of this invention with a mammal for aperiod of time sufficient to yield a metabolic product thereof.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities.

Mixtures of diastereomers may separate under high resolution analyticalprocedures such as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,“Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., NewYork, 1994. The compounds of the invention may contain asymmetric orchiral centers, and therefore exist in different stereoisomeric forms.It is intended that all stereoisomeric forms of the compounds of theinvention, including but not limited to, diastereomers, enantiomers andatropisomers, as well as mixtures thereof such as racemic mixtures, formpart of the present invention. Many organic compounds exist in opticallyactive forms, i.e., they have the ability to rotate the plane ofplane-polarized light. In describing an optically active compound, theprefixes D and L, or R and S, are used to denote the absoluteconfiguration of the molecule about its chiral center(s). The prefixes dand l or (+) and (−) are employed to designate the sign of rotation ofplane-polarized light by the compound, with (−) or l meaning that thecompound is levorotatory. A compound prefixed with (+) or d isdextrorotatory. For a given chemical structure, these stereoisomers areidentical except that they are mirror images of one another. A specificstereoisomer may also be referred to as an enantiomer, and a mixture ofsuch isomers is often called an enantiomeric mixture. A 50:50 mixture ofenantiomers is referred to as a racemic mixture or a racemate, which mayoccur where there has been no stereoselection or stereospecificity in achemical reaction or process. The terms “racemic mixture” and “racemate”refer to an equimolar mixture of two enantiomeric species, devoid ofoptical activity. Enantiomers may be separated from a racemic mixture bya chiral separation method, such as supercritical fluid chromatography(SFC). Assignment of configuration at chiral centers in separatedenantiomers may be tentative, and depicted in Table 1 structures forillustrative purposes, while stereochemical determination awaits, suchas x-ray crystallographic data.

The term “tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies which are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

The term “pharmaceutically acceptable salts” denotes salts which are notbiologically or otherwise undesirable. Pharmaceutically acceptable saltsinclude both acid and base addition salts. The phrase “pharmaceuticallyacceptable” indicates that the substance or composition must becompatible chemically and/or toxicologically, with the other ingredientscomprising a formulation, and/or the mammal being treated therewith.

The term “pharmaceutically acceptable acid addition salt” denotes thosepharmaceutically acceptable salts formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,carbonic acid, phosphoric acid, and organic acids selected fromaliphatic, cycloaliphatic, aromatic, aryl-aliphatic, heterocyclic,carboxylic, and sulfonic classes of organic acids such as formic acid,acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid,pyruvic acid, oxalic acid, malic acid, maleic acid, malonic acid,succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid,ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamicacid, mandelic acid, embonic acid, phenylacetic acid, methanesulfonicacid “mesylate”, ethanesulfonic acid, p-toluenesulfonic acid, andsalicyclic acid.

The term “pharmaceutically acceptable base addition salt” denotes thosepharmaceutically acceptable salts formed with an organic or inorganicbase. Examples of acceptable inorganic bases include sodium, potassium,ammonium, calcium, magnesium, iron, zinc, copper, manganese, andaluminum salts. Salts derived from pharmaceutically acceptable organicnontoxic bases includes salts of primary, secondary, and tertiaryamines, substituted amines including naturally occurring substitutedamines, cyclic amines and basic ion exchange resins, such asisopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,methylglucamine, theobromine, purines, piperazine, piperidine,N-ethylpiperidine, and polyamine resins

A “solvate” refers to an association or complex of one or more solventmolecules and a compound of the invention. Examples of solvents thatform solvates include, but are not limited to, water, isopropanol,ethanol, methanol, DMSO, ethylacetate, acetic acid, and ethanolamine.

The term “EC₅₀” is the half maximal effective concentration” and denotesthe plasma concentration of a particular compound required for obtaining50% of the maximum of a particular effect in vivo.

The term “Ki” is the inhibition constant and denotes the absolutebinding affinity of a particular inhibitor to a receptor. It is measuredusing competition binding assays and is equal to the concentration wherethe particular inhibitor would occupy 50% of the receptors if nocompeting ligand (e.g. a radioligand) was present. Ki values can beconverted logarithmically to pKi values (−log Ki), in which highervalues indicate exponentially greater potency.

The term “IC₅₀” is the half maximal inhibitory concentration and denotesthe concentration of a particular compound required for obtaining 50%inhibition of a biological process in vitro. IC₅₀ values can beconverted logarithmically to pIC₅₀ values (−log IC₅₀), in which highervalues indicate exponentially greater potency. The IC₅₀ value is not anabsolute value but depends on experimental conditions e.g.concentrations employed, and can be converted to an absolute inhibitionconstant (Ki) using the Cheng-Prusoff equation (Biochem. Pharmacol.(1973) 22:3099). Other percent inhibition parameters, such as IC₇₀,IC₉₀, etc., may be calculated.

The terms “compound of this invention,” and “compounds of the presentinvention” and “compounds of Formula I” include compounds of Formulas Iand stereoisomers, geometric isomers, tautomers, solvates, metabolites,and pharmaceutically acceptable salts and prodrugs thereof.

Any formula or structure given herein, including Formula I compounds, isalso intended to represent hydrates, solvates, and polymorphs of suchcompounds, and mixtures thereof.

Any formula or structure given herein, including Formula I compounds, isalso intended to represent unlabeled forms as well as isotopicallylabeled forms of the compounds. Isotopically labeled compounds havestructures depicted by the formulas given herein except that one or moreatoms are replaced by an atom having a selected atomic mass or massnumber. Examples of isotopes that can be incorporated into compounds ofthe invention include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorous, fluorine, and chlorine, such as, but not limited to 2H(deuterium, D), 3H (tritium), 11C, 13C, 14C, 15N, 18F, 31P, 32P, 35S,36Cl, and 125I. Various isotopically labeled compounds of the presentinvention, for example those into which radioactive isotopes such as 3H,13C, and 14C are incorporated. Such isotopically labeled compounds maybe useful in metabolic studies, reaction kinetic studies, detection orimaging techniques, such as positron emission tomography (PET) orsingle-photon emission computed tomography (SPECT) including drug orsubstrate tissue distribution assays, or in radioactive treatment ofpatients. Deuterium labeled or substituted therapeutic compounds of theinvention may have improved DMPK (drug metabolism and pharmacokinetics)properties, relating to distribution, metabolism, and excretion (ADME).Substitution with heavier isotopes such as deuterium may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample increased in vivo half-life or reduced dosage requirements. An18F labeled compound may be useful for PET or SPECT studies.Isotopically labeled compounds of this invention and prodrugs thereofcan generally be prepared by carrying out the procedures disclosed inthe schemes or in the examples and preparations described below bysubstituting a readily available isotopically labeled reagent for anon-isotopically labeled reagent. Further, substitution with heavierisotopes, particularly deuterium (i.e., 2H or D) may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample increased in vivo half-life or reduced dosage requirements or animprovement in therapeutic index. It is understood that deuterium inthis context is regarded as a substituent in the compound of the formula(I). The concentration of such a heavier isotope, specificallydeuterium, may be defined by an isotopic enrichment factor. In thecompounds of this invention any atom not specifically designated as aparticular isotope is meant to represent any stable isotope of thatatom. Unless otherwise stated, when a position is designatedspecifically as “H” or “hydrogen”, the position is understood to havehydrogen at its natural abundance isotopic composition. Accordingly, inthe compounds of this invention any atom specifically designated as adeuterium (D) is meant to represent deuterium.

Heteroaryl Pyridone and Aza-Pyridone Amide Compounds with ElectrophilicFunctionality

The present invention provides heteroaryl pyridone and aza-pyridoneamide compounds of Formula I, including Formulas Ia-Id, andpharmaceutical formulations thereof, which are potentially useful in thetreatment of diseases, conditions and/or disorders modulated by Btk.

Formula I compounds have the structure:

or stereoisomers, tautomers, or pharmaceutically acceptable saltsthereof, wherein:

X¹ is CR¹ or N;

X² is CR² or N;

X³ is CR³ or N;

R¹, R² and R³ are independently selected from H, F, Cl, CN, —NH₂,—NHCH₃, —N(CH₃)₂, —OH, —OCH₃, —OCH₂CH₃, —OCH₂CH₂OH, and C₁-C₃ alkyl;

X⁴, X⁵, X⁶, and X⁷ are independently selected from CH and N;

Y¹ and Y² are independently selected from CH and N;

Z is O or NR, where R is H or C₁-C₃ alkyl;

Q is selected from the groups having the structure:

where R⁴ is selected from —CH═CH₂, —C(CH₃)═CH₂, —C(CN)═CH₂, —C≡CCH₃, and—C≡CH; and R⁵ is selected from H and C₁-C₃ alkyl;

R^(6a), R^(6b), R^(7a), and R^(7b) are independently selected from H, F,Cl, CN, —NH₂, —NHCH₃, —N(CH₃)₂, —OH, —OCH₃, —OCH₂CH₃, —OCH₂CH₂OH, andC₁-C₃ alkyl;

or R^(6a) and R^(7a) form a five-, six-, or seven-membered carbocyclylor heterocyclyl ring;

or R⁵ and R^(6a) form a five-, six-, or seven-membered heterocyclylring;

or if Z is nitrogen, then Z and R^(7a), or Z and R^(6a) form a five-,six-, or seven-membered heterocyclyl ring;

R⁸ is selected from H, F, Cl, CN, —CH₂OH, —CH(CH₃)OH, —C(CH₃)₂OH,—CH(CF₃)OH, —CH₂F, —CHF₂, —CH₂CHF₂, —CF₃, —C(O)NH₂, —C(O)NHCH₃,—C(O)N(CH₃)₂, —NH₂, —NHCH₃, —N(CH₃)₂, —NHC(O)CH₃, —OH, —OCH₃, —OCH₂CH₃,—OCH₂CH₂OH, cyclopropyl, cyclopropylmethyl, 1-hydroxycyclopropyl,imidazolyl, pyrazolyl, 3-hydroxy-oxetan-3-yl, oxetan-3-yl, andazetidin-1-yl;

R⁹ is selected from the structures:

where the wavy line indicates the site of attachment; and

where alkyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl areoptionally substituted with one or more groups independently selectedfrom F, Cl, Br, I, —CN, —CH₃, —CH₂CH₃, —CH(CH₃)₂, —CH₂CH(CH₃)₂, —CH₂OH,—CH₂OCH₃, —CH₂CH₂OH, —C(CH₃)₂OH, —CH(OH)CH(CH₃)₂, —C(CH₃)₂CH₂OH,—CH₂CH₂SO₂CH₃, —CH₂OP(O)(OH)₂, —CH₂F, —CHF₂, —CF₃, —CH₂CF₃, —CH₂CHF₂,—CH(CH₃)CN, —C(CH₃)₂CN, —CH₂CN, —CO₂H, —COCH₃, —CO₂CH₃, —CO₂C(CH₃)₃,—COCH(OH)CH₃, —CONH₂, —CONHCH₃, —CON(CH₃)₂, —C(CH₃)₂CONH₂, —NH₂, —NHCH₃,—N(CH₃)₂, —NHCOCH₃, —N(CH₃)COCH₃, —NHS(O)₂CH₃, —N(CH₃)C(CH₃)₂CONH₂,—N(CH₃)CH₂CH₂S(O)₂CH₃, —NO₂, ═O, —OH, —OCH₃, —OCH₂CH₃, —OCH₂CH₂OCH₃,—OCH₂CH₂OH, —OCH₂CH₂N(CH₃)₂, —OP(O)(OH)₂, —S(O)₂N(CH₃)₂, —SCH₃,—S(O)₂CH₃, —S(O)₃H, cyclopropyl, oxetanyl, azetidinyl,1-methylazetidin-3-yl)oxy, N-methyl-N-oxetan-3-ylamino,azetidin-1-ylmethyl, pyrrolidin-1-yl, and morpholino.

Exemplary embodiments of Formula I compounds include wherein:

X¹ is N;

X² is N;

X³ is N;

X¹ and X³ are N, X¹ and X² are N, or X² and X³ are N;

X¹ and X³ are CH, and X² is CF;

X⁴ is N;

X⁴ and X⁵ are N;

Y¹ is CH and Y² is N;

Y¹ is N and Y² is CH;

Y¹ and Y² are each CH;

R⁴ is —CH═CH₂;

R⁵ is H or —CH₃;

R^(6a), R^(6b), R^(7a), and R^(7b) are H; or

R⁸ is —CH₂OH.

Exemplary embodiments of Formula I compounds include wherein R⁹ isselected from:

Exemplary embodiments of Formula I compounds include compounds ofFormulas Ia-d:

Exemplary embodiments of Formula I compounds include wherein the group:

-   -   is selected from:

Exemplary embodiments of Formula I compounds include wherein Z isnitrogen, and and R^(6a) form a five-, six-, or seven-memberedheterocyclyl ring, and the heterocyclyl ring is pyrrolidinyl.

Exemplary embodiments of Formula I compounds include compounds fromTable 1.

Although the scope of the invention is not limited by any particularmechanism of action, binding properties, or interaction of the compoundsof the invention with a kinase target, such as Btk, the electrophilicfunctionality of Formula I compounds may form a covalent bond with Btk.The covalent bond so formed may be reversibly or irreversibly formed.

The Formula I compounds of the invention may contain asymmetric orchiral centers, and therefore exist in different stereoisomeric forms.It is intended that all stereoisomeric forms of the compounds of theinvention, including but not limited to, diastereomers, enantiomers andatropisomers, as well as mixtures thereof such as racemic mixtures, formpart of the present invention.

In addition, the present invention embraces all diastereomers, includingcis-trans (geometric) and conformational isomers. For example, if aFormula I compound incorporates a double bond or a fused ring, the cis-and trans-forms, as well as mixtures thereof, are embraced within thescope of the invention.

In the structures shown herein, where the stereochemistry of anyparticular chiral atom is not specified, then all stereoisomers arecontemplated and included as the compounds of the invention. Wherestereochemistry is specified by a solid wedge or dashed linerepresenting a particular configuration, then that stereoisomer is sospecified and defined.

The compounds of the present invention may exist in unsolvated as wellas solvated forms with pharmaceutically acceptable solvents such aswater, ethanol, and the like, and it is intended that the inventionembrace both solvated and unsolvated forms.

The compounds of the present invention may also exist in differenttautomeric forms, and all such forms are embraced within the scope ofthe invention. The term “tautomer” or “tautomeric form” refers tostructural isomers of different energies which are interconvertible viaa low energy barrier. For example, proton tautomers (also known asprototropic tautomers) include interconversions via migration of aproton, such as keto-enol and imine-enamine isomerizations. Valencetautomers include interconversions by reorganization of some of thebonding electrons.

Biological Evaluation

The relative efficacies of Formula I compounds as inhibitors of anenzyme activity (or other biological activity) can be established bydetermining the concentrations at which each compound inhibits theactivity to a predefined extent and then comparing the results.Typically, the preferred determination is the concentration thatinhibits 50% of the activity in a biochemical assay, i.e., the 50%inhibitory concentration or “IC₅₀”. Determination of IC₅₀ values can beaccomplished using conventional techniques known in the art. In general,an IC₅₀ can be determined by measuring the activity of a given enzyme inthe presence of a range of concentrations of the inhibitor under study.The experimentally obtained values of enzyme activity then are plottedagainst the inhibitor concentrations used. The concentration of theinhibitor that shows 50% enzyme activity (as compared to the activity inthe absence of any inhibitor) is taken as the IC₅₀ value. Analogously,other inhibitory concentrations can be defined through appropriatedeterminations of activity. For example, in some settings it can bedesirable to establish a 90% inhibitory concentration, i.e., IC₉₀, etc.

Formula I compounds were tested by biochemical Btk, Kinase assays(Example 901).

A general procedure for a standard cellular Btk, Kinase Assay that canbe used to test Formula I compounds is a Ramos Cell Btk Assay (Example902).

A standard cellular B-cell proliferation assay can be used to testFormula I compounds with B-cells purified from spleen of Balb/c mice(Example 903).

A standard T cell proliferation assay can be used to test Formula Icompounds with T-cells purified from spleen of Balb/c mice (Example904).

A CD86 Inhibition assay can be conducted on Formula I compounds for theinhibition of B cell activity using total mouse splenocytes purifiedfrom spleens of 8-16 week old Balb/c mice (Example 905).

A B-ALL Cell Survival Assay can be conducted on Formula I compounds tomeasure the number of viable B-ALL cells in culture (Example 906).

A CD69 Whole Blood Assay can be conducted on Formula I compounds todetermine the ability of compounds to inhibit the production of CD69 byB lymphocytes in human whole blood activated by crosslinking surface IgMwith goat F(ab′)2 anti-human IgM (Example 907). CD69 is a type II C-typelectin involved in lymphocyte migration and cytokine secretion. CD69expression represents one of the earliest available indicators ofleukocyte activation and its rapid induction occurs throughtranscriptional activation (Vazquez et al (2009) Jour. of ImmunologyPublished Oct. 19, 2009, doi:10.4049/jimmunol.0900839).Concentration-dependent inhibition of antigen receptor stimulation byselective Btk inhibitors induces cell surface expression of thelymphocyte activation marker CD69 (Honigberg et al (2010) Proc. Natl.Acad. Sci. 107(29):13075-13080). Thus, CD69 inhibition by selective Btkinhibitors may be correlated with therapeutic efficacy of certain B-celldisorders. The CD69 Hu Blood FACS IC70 values are displayed forexemplary Formula I compounds in Tables 1 and 2.

The cytotoxic or cytostatic activity of Formula I exemplary compoundscan be measured by: establishing a proliferating mammalian tumor cellline in a cell culture medium, adding a Formula I compound, culturingthe cells for a period from about 6 hours to about 5 days; and measuringcell viability (Example 908). Cell-based in vitro assays are used tomeasure viability, i.e. proliferation (IC₅₀), cytotoxicity (EC₅₀), andinduction of apoptosis (caspase activation) and may be useful inpredicting clinical efficacy against hematological malignancies andsolid tumors.

The in vitro potency of the combinations of Formula I compounds withchemotherapeutic agents can be measured by the cell proliferation assayof Example 908; the CellTiter-Glo® Luminescent Cell Viability Assay,commercially available from Promega Corp., Madison, Wis. Thishomogeneous assay method is based on the recombinant expression ofColeoptera luciferase (U.S. Pat. No. 5,583,024; U.S. Pat. No. 5,674,713;U.S. Pat. No. 5,700,670) and determines the number of viable cells inculture based on quantitation of the ATP present, an indicator ofmetabolically active cells (Crouch et al (1993) J. Immunol. Meth.160:81-88; U.S. Pat. No. 6,602,677). The CellTiter-Glo® Assay wasconducted in 96 or 384 well format, making it amenable to automatedhigh-throughput screening (HTS) (Cree et al (1995) AntiCancer Drugs6:398-404). The homogeneous assay procedure involves adding the singlereagent (CellTiter-Glo® Reagent) directly to cells cultured inserum-supplemented medium. Cell washing, removal of medium and multiplepipetting steps are not required. The system detects as few as 15cells/well in a 384-well format in 10 minutes after adding reagent andmixing.

The homogeneous “add-mix-measure” format results in cell lysis andgeneration of a luminescent signal proportional to the amount of ATPpresent. The amount of ATP is directly proportional to the number ofcells present in culture. The CellTiter-Glo® Assay generates a“glow-type” luminescent signal, produced by the luciferase reaction,which has a half-life generally greater than five hours, depending oncell type and medium used. Viable cells are reflected in relativeluminescence units (RLU). The substrate, Beetle Luciferin, isoxidatively decarboxylated by recombinant firefly luciferase withconcomitant conversion of ATP to AMP and generation of photons. Theextended half-life eliminates the need to use reagent injectors andprovides flexibility for continuous or batch mode processing of multipleplates. This cell proliferation assay can be used with various multiwellformats, e.g. 96 or 384 well format. Data can be recorded by luminometeror CCD camera imaging device. The luminescence output is presented asrelative light units (RLU), measured over time.

The anti-proliferative efficacy of Formula I exemplary compounds andcombinations with chemotherapeutic agents are measured by theCellTiter-Glo® Assay (Example 908) against certain hematological tumorcell lines. EC₅₀ values are established for the tested compounds andcombinations.

Dosing of Formula I compounds for treating an autoimmune disease can beassessed in a mouse model of rheumatoid arthritis. In this model,arthritis is induced in Balb/c mice by administering anti-collagenantibodies and lipopolysaccharide. See Nandakumar et al. (2003), Am. J.Pathol 163:1827-1837. In another example, dosing of Formula I compoundsfor the treatment of B-cell proliferative disorders can be examined in,e.g., a human-to-mouse xenograft model in which human B-cell lymphomacells (e.g. Ramos cells) are implanted into immunodeficient mice (e.g.,“nude” mice) as described in, e.g., Pagel et al. (2005), Clin Cancer Res11(13):4857-4866.

The therapeutic efficacy of a Formula I compound for one of theforegoing diseases can be optimized during a course of treatment. Forexample, a subject being treated can undergo a diagnostic evaluation tocorrelate the relief of disease symptoms or pathologies to inhibition ofin vivo Btk activity achieved by administering a given dose of Formula Icompound. Cellular assays known in the art can be used to determine invivo activity of Btk in the presence or absence of a Formula I compound.For example, since activated Btk is phosphorylated at tyrosine 223(Y223) and tyrosine 551 (Y551), phospho-specific immunocytochemicalstaining of P-Y223 or P-Y551-positive cells can be used to detect orquantify activation of Btk in a population of cells, for example by FACSanalysis of stained vs. unstained cells (Nisitani et al. (1999), Proc.Natl. Acad. Sci, USA 96:2221-2226). Thus, the amount of the Formula Icompound that is administered to a subject can be increased or decreasedas needed so as to maintain a level of Btk inhibition optimal fortreating the subject's disease state.

Generally, a Formula I compound used in the methods described herein isidentified or characterized in an in vitro assay, e.g., an acellularbiochemical assay or a cellular functional assay. Such assays are usefulto determine an in vitro IC50 for a Formula I compound. For example, anacellular kinase assay can be used to determine Btk activity afterincubation of the kinase in the absence or presence of a range ofconcentrations of a candidate irreversible Btk inhibitor compound. Ifthe a Formula I compound is in fact an irreversible Btk inhibitor, Btkkinase activity will not be recovered by repeat washing withinhibitor-free medium (Smaill, et al. (1999), J. Med. Chem.42(10):1803-1815). Further, covalent complex formation between Btk and acandidate irreversible Btk inhibitor is a useful indicator ofirreversible inhibition of Btk that can be readily determined by anumber of methods known in the art (e.g., mass spectrometry). Forexample, some irreversible Btk-inhibitor compounds can form a covalentbond with Cys 481 of Btk (e.g., via a Michael reaction). Cellularfunctional assays for Btk inhibition include measuring one or morecellular endpoints in response to stimulating a Btk-mediated pathway ina cell line (e.g., BCR activation in Ramos cells) in the absence orpresence of a range of concentrations of a Formula I compound. Usefulendpoints for determining a response to BCR activation include, e.g.,autophosphorylation of Btk, phosphorylation of a Btk target protein(e.g., PLC-.gamma.), and cytoplasmic calcium flux.

Against a panel of kinases (Blk, Bmx, Btk, EGFR, ErbB2, ErbB4, Fgr, Itk,JAK1, JAK2, JAK3, Lck, Lyn, SLK, Src, TEC, and TXK) with a Cys residuein the same location as Btk, Compound 101 showed better selectivitytowards Btk than ibrutinib, measured by % inhibition at 1 μM drugconcentration.

By peptide mapping and LCMS detection, it was determined that ibrutiniband Compound 101 only irreversibly bind to wild type Btk and not to theCys-481 Ser mutant Btk (S481C), indicating that Cys-481 is essential totheir irreversible binding. Compound 102 was determined to be areversible binder to wild type Btk.

Exemplary Formula I compounds in Tables 1 and 2 were made,characterized, and tested for inhibition of Btk according to the methodsof this invention, and have the following structures and correspondingnames (ChemDraw Ultra, Version 9.0.1, and ChemBioDraw, Version 11.0,CambridgeSoft Corp., Cambridge Mass.). Where more than one name isassociated with a Formula I compound or intermediate, the chemicalstructure shall define the compound.

TABLE 1 CD69 BTK Hu Blood LC3K FACS No. Structure IUPAC Name MW (Ki, μM)(IC50, μM) 101

N-{2-[(6-{[5-(2-{4,4- dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca- 2(6),7-dien-10-yl}-3-(hydroxymethyl)pyridin-4- yl)-1-methyl-2-oxo-1,2- dihydropyridin-3-yl]amino}pyridin-2- yl)oxy]ethyl}prop-2-enamide 623.70 0.000792 0.0143102

N-(cyanomethyl)-1-(4-{[5- (2-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^(2,6)] dodeca-2(6),7-dien-10-yl}-3-(hydroxymethyl)pyridin-4- yl)-1-methyl-2-oxo-1,2- dihydropyridin-3-yl]amino}pyrimidin-2- yl)pyrrolidine-3-carboxamide 662.74 0.000864 0.655103

N-[2-[[6-[[5-[5-fluoro-2- (hydroxymethyl)-3-(4-oxo- 6,7,8,9-tetrahydrobenzothiopheno[2, 3-d]pyridazin-3-yl)phenyl]-1-methyl-2-oxo-3- pyridyl]amino]-2- pyridyl]oxy]ethyl]prop-2- enamide642.7 0.0616 104

N-[2-[[6-[[5-[2-(6-tert-butyl- 8-fluoro-1-oxo-phthalazin-2-yl)-3-(hydroxymethyl)-4- pyridyl]-1-methyl-2-oxo-3- pyridyl]amino]-2-pyridyl]oxy]ethyl]prop-2- enamide 639.68 0.000915 0.0242 105

N-[2-[[6-[[5-[5-fluoro-2- (hydroxymethyl)-3-(4-oxo- 6,7,8,9-tetrahydrobenzothiopheno[2, 3-d]pyridazin-3-yl)phenyl]-1-methyl-2-oxo-3- pyridyl]amino]-2- pyridyl]amino]ethyl]prop-2- enamide641.72 0.000248 0.0746 106

N-[2-[[6-[[5-[5-fluoro-2- (hydroxymethyl)-3-(4-oxo- 6,7,8,9-tetrahydrobenzothiopheno[2, 3-d]pyridazin-3-yl)phenyl]-1-methyl-2-oxo-3- pyridyl]amino]-2- pyridyl]oxy]ethyl]but-2- ynamide654.71 0.000306 0.0941

TABLE 2 CD69 BTK Hu Blood LC3K FACS No. Structure IUPAC Name MW (Ki, μM)(IC50 μM) 107

N-[(1S)-2-[[6-[[5-[2- (7,7-dimethyl-4-oxo- 1,2,6,8-tetrahydrocyclopenta[3, 4]pyrrolo[3,5- b]pyrazin-3-yl)-3-(hydroxymethyl)-4- pyridyl]-1-methyl-2- oxo-3-pyridyl]amino]-2-pyridyl]oxy]-1- methyl-ethyl]prop-2- enamide 637.728 0.000612 0.363108

N-[(1S)-2-[[6-[[5-[5- fluoro-2- (hydroxymethyl)-3-(4- oxo-6,7,8,9-tetrahydrobenzothio- pheno[2,3-d]pyridazin-3- yl)phenyl]-1-methyl-2-oxo-3-pyridyl]amino]- 2-pyridyl]oxy]-1- methyl-ethyl]prop-2- enamide656.726 0.00257 0.155 109

N-[2-[[6-[[5-[2-(7,7- dimethyl-4-oxo-1,2,6,8- tetrahydrocyclopenta[3,4]pyrrolo[3,5- b]pyrazin-3-yl)-3- (hydroxymethyl)-4-pyridyl]-1-methyl-2- oxo-3-pyridyl]amino]- 2- pyridyl]amino]ethyl]prop-2-enamide 622.717 0.00433 0.102 110

N-[2-[[6-[[5-[2-(7,7- dimethyl-4-oxo-1,2,6,8- tetrahydrocyclopenta[3,4]pyrrolo[3,5- b]pyrazin-3-yl)-3- (hydroxymethyl)-4-pyridyl]-1-methyl-2- oxo-3-pyridyl]amino]- 2-pyridyl]-methyl-amino]ethyl]prop-2- enamide 636.743 0.000683 0.0643 111

N-[2-[[6-[[5-[5-fluoro- 2-(hydroxymethyl)-3- (4-oxo-6,7,8,9-tetrahydrobenzothio- pheno[2,3-d]pyridazin-3- yl)phenyl]-1-methyl-2-oxo-3-pyridyl]amino]- 2-pyridyl]-methyl- amino]ethyl]prop-2- enamide655.742 0.000494 0.174 112

N-[2-[[6-[[5-[2-(7,7- dimethyl-4-oxo-1,2,6,8- tetrahydrocyclopenta[3,4]pyrrolo[3,5- b]pyrazin-3-yl)-3- (hydroxymethyl)-4-pyridyl]-1-methyl-2- oxo-3-pyridyl]amino]- 2-pyridyl]oxy]ethyl]-N-methyl-prop-2-enamide 637.728 0.00175 0.0229 113

N-[2-[[6-[[5-[5-fluoro- 2-(hydroxymethyl)-3- (4-oxo-6,7,8,9-tetrahydrobenzothio- pheno[2,3-d]pyridazin-3- yl)phenyl]-1-methyl-2-oxo-3-pyridyl]amino]- 2-pyridyl]oxy]ethyl]-N- methyl-prop-2-enamide656.726 0.000146 0.127 114

N-[(3S)-1-[6-[[5-[2- (7,7-dimethyl-4-oxo- 1,2,6,8-tetrahydrocyclopenta[3, 4]pyrrolo[3,5- b]pyrazin-3-yl)-3-(hydroxymethyl)-4- pyridyl]-1-methyl-2- oxo-3-pyridyl]amino]-2-pyridyl]-3- piperidyl]prop-2- enamide 662.781 0.000888 7.1 115

N-[(3S)-1-[6-[[5-[5- fluoro-2- (hydroxymethyl)-3-(4- oxo-6,7,8,9-tetrahydrobenzothio- pheno[2,3-d]pyridazin-3- yl)phenyl]-1-methyl-2-oxo-3-pyridyl]amino]- 2-pyridyl]-3- piperidyl]prop-2- enamide 681.7790.00216 116

N-[2-[[6-[[5-[2-(7,7- dimethyl-4-oxo-1,2,6,8- tetrahydrocyclopenta[3,4]pyrrolo[3,5- b]pyrazin-3-yl)-3- (hydroxymethyl)-4-pyridyl]-1-methyl-2- oxo-3-pyridyl]amino]- 2- pyridyl]oxy]ethyl]but-2-ynamide 635.712 0.00237 0.0146 117

2-cyano-N-[2-[[6-[[5- [5-fluoro-2- (hydroxymethyl)-3-(4- oxo-6,7,8,9-tetrahydrobenzothio- pheno[2,3-d]pyridazin-3- yl)phenyl]-1-methyl-2-oxo-3-pyridyl]amino]- 2- pyridyl]oxy]ethyl]prop- 2-enamide 667.7090.000576 0.286 118

2-cyano-N-[2-[[6-[[5- [2-(7,7-dimethyl-4-oxo- 1,2,6,8-tetrahydrocyclopenta[3, 4]pyrrolo[3,5- b]pyrazin-3-yl)-3-(hydroxymethyl)-4- pyridyl]-1-methyl-2- oxo-3-pyridyl]amino]- 2-pyridyl]oxy]ethyl]prop- 2-enamide 648.711 0.0017 0.171

Compound 119 was prepared as the reduced, saturated-double bond analogof compound 101.

Compound 101 was shown to bind covalently to BTK via mass spectroscopyand x-ray crystal structure analyses. Compound 119 (BTK LC3K(Ki=0.000963 μM) would not be expected to bind covalently to BTK.

Administration of Formula I Compounds

The compounds of the invention may be administered by any routeappropriate to the condition to be treated. Suitable routes includeoral, parenteral (including subcutaneous, intramuscular, intravenous,intraarterial, intradermal, intrathecal and epidural), transdermal,rectal, nasal, topical (including buccal and sublingual), vaginal,intraperitoneal, intrapulmonary and intranasal. For localimmunosuppressive treatment, the compounds may be administered byintralesional administration, including perfusing or otherwisecontacting the graft with the inhibitor before transplantation. It willbe appreciated that the preferred route may vary with for example thecondition of the recipient. Where the compound is administered orally,it may be formulated as a pill, capsule, tablet, etc. with apharmaceutically acceptable carrier or excipient. Where the compound isadministered parenterally, it may be formulated with a pharmaceuticallyacceptable parenteral vehicle and in a unit dosage injectable form, asdetailed below.

A dose to treat human patients may range from about 10 mg to about 1000mg of Formula I compound. A typical dose may be about 100 mg to about300 mg of the compound. A dose may be administered once a day (QID),twice per day (BID), or more frequently, depending on thepharmacokinetic and pharmacodynamic properties, including absorption,distribution, metabolism, and excretion of the particular compound. Inaddition, toxicity factors may influence the dosage and administrationregimen. When administered orally, the pill, capsule, or tablet may beingested daily or less frequently for a specified period of time. Theregimen may be repeated for a number of cycles of therapy.

Methods of Treatment with Formula I Compounds

The methods described herein include administering to a subject in needa composition containing a therapeutically effective amount of a FormulaI compound. Without being bound by theory, the diverse roles played byBtk signaling in various hematopoietic cell functions, e.g., B-cellreceptor activation, suggests that small molecule Btk inhibitors areuseful for reducing the risk of or treating a variety of diseasesaffected by or affecting many cell types of the hematopoietic lineageincluding, e.g., autoimmune diseases, heteroimmune conditions ordiseases, inflammatory diseases, cancer (e.g., B-cell proliferativedisorders), and thromboembolic disorders. Further, the irreversible Btkinhibitor compounds described herein may be used to inhibit a smallsubset of other tyrosine kinases that share homology with Btk by havinga cysteine residue (including a Cys 481 residue) that can form acovalent bond with the irreversible inhibitor.

Formula I compounds of the present invention are useful for treating ahuman or animal patient suffering from a disease or disorder arisingfrom abnormal cell growth, function or behavior associated with Btk suchas an immune disorder, cardiovascular disease, viral infection,inflammation, a metabolism/endocrine disorder or a neurologicaldisorder, may thus be treated by a method comprising the administrationthereto of a compound of the present invention as defined above. A humanor animal patient suffering from cancer may also be treated by a methodcomprising the administration thereto of a compound of the presentinvention as defined above. The condition of the patient may thereby beimproved or ameliorated.

Formula I compounds may be useful for in vitro, in situ, and in vivodiagnosis or treatment of mammalian cells, organisms, or associatedpathological conditions, such as systemic and local inflammation,immune-inflammatory, or autoimmune diseases such as rheumatoidarthritis, immune suppression, organ transplant rejection, allergies,ulcerative colitis, Crohn's disease, dermatitis, asthma, systemic lupuserythematosus, extra-renal lupus, Sjögren's Syndrome, multiplesclerosis, scleroderma/systemic sclerosis, idiopathic thrombocytopenicpurpura (ITP), anti-neutrophil cytoplasmic antibodies (ANCA) vasculitis,chronic obstructive pulmonary disease (COPD), psoriasis, psoriaticarthritis, osteoarthritis, Still's disease, juvenile arthritis,diabetes, myasthenia gravis, Hashimoto's thyroiditis, Ord's thyroiditis,Graves' disease Sjogren's syndrome, multiple sclerosis, Guillain-Barresyndrome, acute disseminated encephalomyelitis, Addison's disease,opsoclonus-myoclonus syndrome, ankylosing spondylitisis,antiphospholipid antibody syndrome, aplastic anemia, autoimmunehepatitis, coeliac disease, Goodpasture's syndrome, idiopathicthrombocytopenic purpura, optic neuritis, scleroderma, primary biliarycirrhosis, Reiter's syndrome, Takayasu's arteritis, temporal arteritis,warm autoimmune hemolytic anemia, Wegener's granulomatosis, alopeciauniversalis, Behcets disease, chronic fatigue, dysautonomnia,endometriosis, interstitial cystitis, neuromyotonia, scleroderma, andvulvodynia.

Methods of the invention also include treating such diseases asarthritic diseases, such as rheumatoid arthritis, monoarticulararthritis, osteoarthritis, gouty arthritis, spondylitis; Behcet disease;sepsis, septic shock, endotoxic shock, gram negative sepsis, grampositive sepsis, and toxic shock syndrome; multiple organ injurysyndrome secondary to septicemia, trauma, or hemorrhage; ophthalmicdisorders such as allergic conjunctivitis, vernal conjunctivitis,uveitis, and thyroid-associated ophthalmopathy; eosinophilic granuloma;pulmonary or respiratory disorders such as asthma, chronic bronchitis,allergic rhinitis, ARDS, chronic pulmonary inflammatory disease (e.g.,chronic obstructive pulmonary disease), silicosis, pulmonarysarcoidosis, pleurisy, alveolitis, vasculitis, emphysema, pneumonia,bronchiectasis, and pulmonary oxygen toxicity; reperfusion injury of themyocardium, brain, or extremities; fibrosis such as cystic fibrosis;keloid formation or scar tissue formation; atherosclerosis; autoimmunediseases, such as systemic lupus erythematosus (SLE), autoimmunethyroiditis, multiple sclerosis, some forms of diabetes, and Reynaud'ssyndrome; and transplant rejection disorders such as GVHD and allograftrejection; chronic glomerulonephritis; inflammatory bowel diseases suchas chronic inflammatory bowel disease (CIBD), Crohn's disease,ulcerative colitis, and necrotizing enterocolitis; inflammatorydermatoses such as contact dermatitis, atopic dermatitis, psoriasis, orurticaria; fever and myalgias due to infection; central or peripheralnervous system inflammatory disorders such as meningitis, encephalitis,and brain or spinal cord injury due to minor trauma; Sjogren's syndrome;diseases involving leukocyte diapedesis; alcoholic hepatitis; bacterialpneumonia; antigen-antibody complex mediated diseases; hypovolemicshock; Type I diabetes mellitus; acute and delayed hypersensitivity;disease states due to leukocyte dyscrasia and metastasis; thermalinjury; granulocyte transfusion-associated syndromes; andcytokine-induced toxicity.

In yet other embodiments, the methods described herein can be used totreat a cancer, e.g., B-cell proliferative disorders, which include, butare not limited to diffuse large B cell lymphoma, follicular lymphoma,chronic lymphocytic lymphoma, chronic lymphocytic leukemia, B-cellprolymphocytic leukemia, lymphoplasmacytic lymphoma/Waldenstrommacroglobulinemia, splenic marginal zone lymphoma, plasma cell myeloma,plasmacytoma, extranodal marginal zone B cell lymphoma, nodal marginalzone B cell lymphoma, mantle cell lymphoma, mediastinal (thymic) large Bcell lymphoma, intravascular large B cell lymphoma, primary effusionlymphoma, burkitt lymphoma/leukemia, and lymphomatoid granulomatosis.

Methods of the invention also include treating solid tumors andhematological malignancies. Cancer types which may be treated withFormula I compounds include breast, ovary, cervix, prostate, testis,genitourinary tract, esophagus, larynx, glioblastoma, neuroblastoma,stomach, skin, keratoacanthoma, lung, epidermoid carcinoma, large cellcarcinoma, non-small cell lung carcinoma (NSCLC), small cell carcinoma,lung adenocarcinoma, bone, colon, adenoma, pancreas, adenocarcinoma,thyroid, follicular carcinoma, undifferentiated carcinoma, papillarycarcinoma, seminoma, melanoma, sarcoma, bladder carcinoma, livercarcinoma and biliary passages, kidney carcinoma, pancreatic, myeloiddisorders, lymphoma, hairy cells, buccal cavity, naso-pharyngeal,pharynx, lip, tongue, mouth, small intestine, colon-rectum, largeintestine, rectum, brain and central nervous system, Hodgkin's,leukemia, bronchus, thyroid, liver and intrahepatic bile duct,hepatocellular, gastric, glioma/glioblastoma, endometrial, melanoma,kidney and renal pelvis, urinary bladder, uterine corpus, uterinecervix, multiple myeloma, acute myelogenous leukemia, chronicmyelogenous leukemia, lymphocytic leukemia, chronic lymphoid leukemia(CLL), myeloid leukemia, oral cavity and pharynx, non-Hodgkin lymphoma,melanoma, and villous colon adenoma.

The methods of the invention can have utility in treating subjects whoare or can be subject to reperfusion injury, i.e., injury resulting fromsituations in which a tissue or organ experiences a period of ischemiafollowed by reperfusion. The term “ischemia” refers to localized tissueanemia due to obstruction of the inflow of arterial blood. Transientischemia followed by reperfusion characteristically results inneutrophil activation and transmigration through the endothelium of theblood vessels in the affected area. Accumulation of activatedneutrophils in turn results in generation of reactive oxygenmetabolites, which damage components of the involved tissue or organ.This phenomenon of “reperfusion injury” is commonly associated withconditions such as vascular stroke (including global and focalischemia), hemorrhagic shock, myocardial ischemia or infarction, organtransplantation, and cerebral vasospasm. To illustrate, reperfusioninjury occurs at the termination of cardiac bypass procedures or duringcardiac arrest when the heart, once prevented from receiving blood,begins to reperfuse. It is expected that inhibition of Btk activity mayresult in reduced amounts of reperfusion injury in such situations.

Symptoms, diagnostic tests, and prognostic tests for each of theabove-mentioned conditions are known in the art. See, e.g., Harrison'sPrinciples of Internal Medicine,” 16th ed., 2004, The McGraw-HillCompanies, Inc. Dey et al. (2006), Cytojournal 3(24), and the “RevisedEuropean American Lymphoma” (REAL) classification system (see, e.g., thewebsite maintained by the National Cancer Institute). A number of animalmodels of are useful for establishing a range of therapeuticallyeffective doses of Formula I compounds for treating any of the foregoingdiseases

Pharmaceutical Formulations

In order to use a compound of this invention for the therapeutictreatment of mammals including humans, it is normally formulated inaccordance with standard pharmaceutical practice as a pharmaceuticalcomposition. According to this aspect of the invention there is provideda pharmaceutical composition comprising a compound of this invention inassociation with a pharmaceutically acceptable diluent or carrier.

The pharmaceutical formulations described herein include, but are notlimited to, aqueous liquid dispersions, self-emulsifying dispersions,solid solutions, liposomal dispersions, aerosols, solid dosage forms,powders, immediate release formulations, controlled releaseformulations, fast melt formulations, tablets, capsules, pills, delayedrelease formulations, extended release formulations, pulsatile releaseformulations, multiparticulate formulations, and mixed immediate andcontrolled release formulations.

Pharmaceutical compositions including a compound described herein may bemanufactured in a conventional manner, such as, by way of example only,by means of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, spray-drying, encapsulating, entrapping orcompression processes, pan coating, melt granulation, granulation,fluidized bed spray drying or coating (e.g., wurster coating),tangential coating, top spraying, tableting, extruding and the like. Atypical formulation is prepared by mixing a compound of the presentinvention and a carrier, diluent or excipient. Suitable carriers,diluents and excipients are well known to those skilled in the art andinclude materials such as carbohydrates, waxes, water soluble and/orswellable polymers, hydrophilic or hydrophobic materials, gelatin, oils,solvents, water and the like. The particular carrier, diluent orexcipient used will depend upon the means and purpose for which thecompound of the present invention is being applied. Solvents aregenerally selected based on solvents recognized by persons skilled inthe art as safe (GRAS) to be administered to a mammal. In general, safesolvents are non-toxic aqueous solvents such as water and othernon-toxic solvents that are soluble or miscible in water. Suitableaqueous solvents include water, ethanol, propylene glycol, polyethyleneglycols (e.g., PEG 400, PEG 300), etc. and mixtures thereof. Theformulations may also include one or more buffers, binders, stabilizingagents, antifoaming agents, surfactants, wetting agents, lubricatingagents, emulsifiers, suspending agents, preservatives, antioxidants,opaquing agents, glidants, processing aids, colorants, sweeteners,perfuming agents, flavoring agents and other known additives to providea stable or elegant presentation of the drug (i.e., a compound of thepresent invention or pharmaceutical composition thereof) or aid in themanufacturing of the pharmaceutical product (i.e., medicament).

Binders impart cohesive qualities and include, e.g., alginic acid andsalts thereof; cellulose derivatives such as carboxymethylcellulose,methylcellulose (e.g., Methocel®), hydroxypropylmethylcellulose,hydroxyethylcellulose, hydroxypropylcellulose (e.g., Klucel®),ethylcellulose (e.g., Ethocel®), and microcrystalline cellulose (e.g.,Avicel®); microcrystalline dextrose; amylose; magnesium aluminumsilicate; polysaccharide acids; bentonites; gelatin;polyvinylpyrrolidone/vinyl acetate copolymer; crosspovidone; povidone;starch; pregelatinized starch; tragacanth, dextrin, a sugar, such assucrose (e.g., Dipac®), glucose, dextrose, molasses, mannitol, sorbitol,xylitol (e.g., Xylitab®), and lactose; a natural or synthetic gum suchas acacia, tragacanth, ghatti gum mucilage of isapol husks,polyvinylpyrrolidone (e.g., Polyvidone® CL, Kollidon® CL, Polyplasdone®XL-10), larch arabogalactan, Veegum®, polyethylene glycol, waxes, sodiumalginate, and the like. In general, binder levels of 20-70% are used inpowder-filled gelatin capsule formulations. Binder usage level in tabletformulations varies whether direct compression, wet granulation, rollercompaction, or usage of other excipients such as fillers which itselfcan act as moderate binder. Formulators skilled in art can determine thebinder level for the formulations, but binder usage level of up to 70%in tablet formulations is common.

The formulations may be prepared using conventional dissolution andmixing procedures. For example, the bulk drug substance (i.e., compoundof the present invention or stabilized form of the compound (e.g.,complex with a cyclodextrin derivative or other known complexationagent) is dissolved in a suitable solvent in the presence of one or moreof the excipients described above. The compound of the present inventionis typically formulated into pharmaceutical dosage forms to provide aneasily controllable dosage of the drug and to enable patient compliancewith the prescribed regimen.

The pharmaceutical composition (or formulation) for application may bepackaged in a variety of ways depending upon the method used foradministering the drug. Generally, an article for distribution includesa container having deposited therein the pharmaceutical formulation inan appropriate form. Suitable containers are well known to those skilledin the art and include materials such as bottles (plastic and glass),sachets, ampoules, plastic bags, metal cylinders, and the like. Thecontainer may also include a tamper-proof assemblage to preventindiscreet access to the contents of the package. In addition, thecontainer has deposited thereon a label that describes the contents ofthe container. The label may also include appropriate warnings.

Pharmaceutical formulations of the compounds of the present inventionmay be prepared for various routes and types of administration. Forexample, a compound of Formula I having the desired degree of purity mayoptionally be mixed with pharmaceutically acceptable diluents, carriers,excipients or stabilizers (Remington's Pharmaceutical Sciences (1980)16th edition, Osol, A. Ed.), in the form of a lyophilized formulation,milled powder, or an aqueous solution. Formulation may be conducted bymixing at ambient temperature at the appropriate pH, and at the desireddegree of purity, with physiologically acceptable carriers, i.e.,carriers that are non-toxic to recipients at the dosages andconcentrations employed. The pH of the formulation depends mainly on theparticular use and the concentration of compound, but may range fromabout 3 to about 8. Formulation in an acetate buffer at pH 5 is asuitable embodiment.

The compound ordinarily can be stored as a solid composition, alyophilized formulation or as an aqueous solution.

The pharmaceutical compositions of the invention will be formulated,dosed and administered in a fashion, i.e., amounts, concentrations,schedules, course, vehicles and route of administration, consistent withgood medical practice. Factors for consideration in this context includethe particular disorder being treated, the particular mammal beingtreated, the clinical condition of the individual patient, the cause ofthe disorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The “therapeutically effective amount”of the compound to be administered will be governed by suchconsiderations, and is the minimum amount necessary to ameliorate, ortreat the hyperproliferative disorder.

As a general proposition, the initial pharmaceutically effective amountof the inhibitor administered parenterally per dose will be in the rangeof about 0.01-100 mg/kg, namely about 0.1 to 20 mg/kg of patient bodyweight per day, with the typical initial range of compound used being0.3 to 15 mg/kg/day.

Acceptable diluents, carriers, excipients and stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate and other organic acids; antioxidantsincluding ascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).Suitable carriers for use in the solid dosage forms described hereininclude, but are not limited to, acacia, gelatin, colloidal silicondioxide, calcium glycerophosphate, calcium lactate, maltodextrin,glycerine, magnesium silicate, sodium caseinate, soy lecithin, sodiumchloride, tricalcium phosphate, dipotassium phosphate, sodium stearoyllactylate, carrageenan, monoglyceride, diglyceride, pregelatinizedstarch, hydroxypropylmethylcellulose, hydroxypropylmethylcelluloseacetate stearate, sucrose, microcrystalline cellulose, lactose, mannitoland the like. Suitable filling agents for use in the solid dosage formsdescribed herein include, but are not limited to, lactose, calciumcarbonate, calcium phosphate, dibasic calcium phosphate, calciumsulfate, microcrystalline cellulose, cellulose powder, dextrose,dextrates, dextran, starches, pregelatinized starch,hydroxypropylmethycellulose (HPMC), hydroxypropylmethycellulosephthalate, hydroxypropylmethylcellulose acetate stearate (HPMCAS),sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride,polyethylene glycol, and the like.

The active pharmaceutical ingredients may be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton,Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975;Libetman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms,Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms andDrug Delivery Systems, Seventh Ed. (Lippincott Williams & Willins 1999).

Sustained-release preparations of compounds of Formula I may beprepared. Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing acompound of Formula I, which matrices are in the form of shapedarticles, e.g., films, or microcapsules. Examples of sustained-releasematrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate) and poly-D-(−)-3-hydroxybutyric acid.

The formulations include those suitable for the administration routesdetailed herein. The formulations may conveniently be presented in unitdosage form and may be prepared by any of the methods well known in theart of pharmacy. Techniques and formulations generally are found inRemington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.).Such methods include the step of bringing into association the activeingredient with the carrier which constitutes one or more accessoryingredients. In general the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

Formulations of a compound of Formula I suitable for oral administrationmay be prepared as discrete units such as pills, capsules, cachets ortablets each containing a predetermined amount of a compound of FormulaI. Compressed tablets may be prepared by compressing in a suitablemachine the active ingredient in a free-flowing form such as a powder orgranules, optionally mixed with a binder, lubricant, inert diluent,preservative, surface active or dispersing agent. Molded tablets may bemade by molding in a suitable machine a mixture of the powdered activeingredient moistened with an inert liquid diluent. The tablets mayoptionally be coated or scored and optionally are formulated so as toprovide slow or controlled release of the active ingredient therefrom.Tablets, troches, lozenges, aqueous or oil suspensions, dispersiblepowders or granules, emulsions, hard or soft capsules, e.g., gelatincapsules, syrups or elixirs may be prepared for oral use. Formulationsof compounds of Formula I intended for oral use may be preparedaccording to any method known to the art for the manufacture ofpharmaceutical compositions and such compositions may contain one ormore agents including sweetening agents, flavoring agents, coloringagents and preserving agents, in order to provide a palatablepreparation. Tablets containing the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipient which are suitable formanufacture of tablets are acceptable. These excipients may be, forexample, inert diluents, such as calcium or sodium carbonate, lactose,calcium or sodium phosphate; granulating and disintegrating agents, suchas maize starch, or alginic acid; binding agents, such as starch,gelatin or acacia; and lubricating agents, such as magnesium stearate,stearic acid or talc. Tablets may be uncoated or may be coated by knowntechniques including microencapsulation to delay disintegration andadsorption in the gastrointestinal tract and thereby provide a sustainedaction over a longer period. For example, a time delay material such asglyceryl monostearate or glyceryl distearate alone or with a wax may beemployed.

“Disintegration agents” or disintegrants facilitate the breakup ordisintegration of a substance. Examples of disintegration agents includea starch, e.g., a natural starch such as corn starch or potato starch, apregelatinized starch such as National 1551 or Amijel®, or sodium starchglycolate such as Promogel® or Explotab®, a cellulose such as a woodproduct, methylcrystalline cellulose, e.g., Avicel®, Elceme®, Emcocel®,Vivacel®, Ming Tia®, and Solka-Floc®, methylcellulose, croscarmellose,or a cross-linked cellulose, such as cross-linked sodiumcarboxymethylcellulose (Ac-Di-Sol®), cross-linkedcarboxymethylcellulose, or cross-linked croscarmellose, a cross-linkedstarch such as sodium starch glycolate, a cross-linked polymer such ascrosspovidone, a cross-linked polyvinylpyrrolidone, alginate such asalginic acid or a salt of alginic acid such as sodium alginate, a claysuch as Veegum® HV (magnesium aluminum silicate), a gum such as agar,guar, locust bean, Karaya, pectin, or tragacanth, sodium starchglycolate, bentonite, a natural sponge, a surfactant, a resin such as acation-exchange resin, citrus pulp, sodium lauryl sulfate, sodium laurylsulfate in combination starch, and the like

Dispersing agents and/or “viscosity modulating agents” include materialsthat control the diffusion and homogeneity of a drug through liquidmedia or a granulation method or blend method. In some embodiments,these agents also facilitate the effectiveness of a coating or erodingmatrix. Exemplary diffusion facilitators/dispersing agents include,e.g., hydrophilic polymers, electrolytes, Tween® 60 or 80, PEG,polyvinylpyrrolidone (PVP; commercially known as Plasdone®), and thecarbohydrate-based dispersing agents such as, for example, hydroxypropylcelluloses (e.g., HPC, H-PC-SL, and HPC-L), hydroxypropylmethylcelluloses (e.g., HPMC K100, RPMC K4M, HPMC K15M, and HPMC K100M),carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate,hydroxypropylmethylcellulose acetate stearate (HPMCAS), noncrystallinecellulose, magnesium aluminum silicate, triethanolamine, polyvinylalcohol (PVA), vinyl pyrrolidone/vinyl acetate copolymer (S630),4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide andformaldehyde (also known as tyloxapol), poloxamers (e.g., Pluronics®,which are block copolymers of ethylene oxide and propylene oxide); andpoloxamines (e.g., Tetronic 908®, also known as Poloxamine®, which is atetrafunctional block copolymer derived from sequential addition ofpropylene oxide and ethylene oxide to ethylenediamine (BASF Corporation,Parsippany, N.J.)), polyvinylpyrrolidone K12, polyvinylpyrrolidone K17,polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30,polyvinylpyrrolidone/vinyl acetate copolymer (S-630), polyethyleneglycol, e.g., the polyethylene glycol can have a molecular weight ofabout 300 to about 6000, or about 3350 to about 4000, or about 7000 toabout 5400, sodium carboxymethylcellulose, methylcellulose,polysorbate-80, sodium alginate, gums, such as, e.g., gum tragacanth andgum acacia, guar gum, xanthans, including xanthan gum, sugars,cellulosics, such as, e.g., sodium carboxymethylcellulose,methylcellulose, sodium carboxymethylcellulose, polysorbate-80, sodiumalginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitanmonolaurate, povidone, carbomers, polyvinyl alcohol (PVA), alginates,chitosans and combinations thereof. Plasticizers such as cellulose ortriethyl cellulose can also be used as dispersing agents. Dispersingagents particularly useful in liposomal dispersions and self-emulsifyingdispersions are dimyristoyl phosphatidyl choline, natural phosphatidylcholine from eggs, natural phosphatidyl glycerol from eggs, cholesteroland isopropyl myristate.

“Lubricants” and “glidants” are compounds that prevent, reduce orinhibit adhesion or friction of materials. Exemplary lubricants include,e.g., stearic acid, calcium hydroxide, talc, sodium stearyl lumerate, ahydrocarbon such as mineral oil, or hydrogenated vegetable oil such ashydrogenated soybean oil (Sterotex®), higher fatty acids and theiralkali-metal and alkaline earth metal salts, such as aluminum, calcium,magnesium, zinc, stearic acid, sodium stearates, glycerol, talc, waxes,Stearowet®, boric acid, sodium benzoate, sodium acetate, sodiumchloride, leucine, a polyethylene glycol (e.g., PEG4000) or amethoxypolyethylene glycol such as Carbowax™, sodium oleate, sodiumbenzoate, glyceryl behenate, polyethylene glycol, magnesium or sodiumlauryl sulfate, colloidal silica such as Syloid®, Cab-O-Sil®, a starchsuch as corn starch, silicone oil, a surfactant, and the like.

For treatment of the eye or other external tissues, e.g., mouth andskin, the formulations are preferably applied as a topical ointment orcream containing the active ingredient(s) in an amount of, for example,0.075 to 20% w/w. When formulated in an ointment, the active ingredientsmay be employed with either a paraffinic or a water-miscible ointmentbase. Alternatively, the active ingredients may be formulated in a creamwith an oil-in-water cream base. If desired, the aqueous phase of thecream base may include a polyhydric alcohol, i.e., an alcohol having twoor more hydroxyl groups such as propylene glycol, butane 1,3-diol,mannitol, sorbitol, glycerol and polyethylene glycol (including PEG 400)and mixtures thereof. The topical formulations may desirably include acompound which enhances absorption or penetration of the activeingredient through the skin or other affected areas. Examples of suchdermal penetration enhancers include dimethyl sulfoxide and relatedanalogs. The oily phase of the emulsions of this invention may beconstituted from known ingredients in a known manner. While the phasemay comprise merely an emulsifier, it desirably comprises a mixture ofat least one emulsifier with a fat or an oil or with both a fat and anoil. Preferably, a hydrophilic emulsifier is included together with alipophilic emulsifier which acts as a stabilizer. It is also preferredto include both an oil and a fat. Together, the emulsifier(s) with orwithout stabilizer(s) make up the so-called emulsifying wax, and the waxtogether with the oil and fat make up the so-called emulsifying ointmentbase which forms the oily dispersed phase of the cream formulations.Emulsifiers and emulsion stabilizers suitable for use in the formulationof the invention include Tween® 60, Span® 80, cetostearyl alcohol,benzyl alcohol, myristyl alcohol, glyceryl mono-stearate and sodiumlauryl sulfate.

Aqueous suspensions of Formula I compounds contain the active materialsin admixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients include a suspending agent, such as sodiumcarboxymethylcellulose, croscarmellose, povidone, methylcellulose,hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone,gum tragacanth and gum acacia, and dispersing or wetting agents such asa naturally occurring phosphatide (e.g., lecithin), a condensationproduct of an alkylene oxide with a fatty acid (e.g., polyoxyethylenestearate), a condensation product of ethylene oxide with a long chainaliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensationproduct of ethylene oxide with a partial ester derived from a fatty acidand a hexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). Theaqueous suspension may also contain one or more preservatives such asethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents and one or more sweetening agents, such as sucroseor saccharin.

The pharmaceutical compositions of compounds of Formula I may be in theform of a sterile injectable preparation, such as a sterile injectableaqueous or oleaginous suspension. This suspension may be formulatedaccording to the known art using those suitable dispersing or wettingagents and suspending agents which have been mentioned above. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a non-toxic parenterally acceptable diluent or solvent,such as a solution in 1,3-butanediol or prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile fixed oils may conventionally be employed as a solventor suspending medium. For this purpose any bland fixed oil may beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid may likewise be used in the preparation ofinjectables.

The amount of active ingredient that may be combined with the carriermaterial to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, atime-release formulation intended for oral administration to humans maycontain approximately 1 to 1000 mg of active material compounded with anappropriate and convenient amount of carrier material which may varyfrom about 5 to about 95% of the total compositions (weight:weight). Thepharmaceutical composition can be prepared to provide easily measurableamounts for administration. For example, an aqueous solution intendedfor intravenous infusion may contain from about 3 to 500 μg of theactive ingredient per milliliter of solution in order that infusion of asuitable volume at a rate of about 30 mL/hr can occur.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the activeingredient. The active ingredient is preferably present in suchformulations in a concentration of about 0.5 to 20% w/w, for exampleabout 0.5 to 10% w/w, for example about 1.5% w/w.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for intrapulmonary or nasal administration have aparticle size for example in the range of 0.1 to 500 microns (includingparticle sizes in a range between 0.1 and 500 microns in incrementsmicrons such as 0.5, 1, 30 microns, 35 microns, etc.), which isadministered by rapid inhalation through the nasal passage or byinhalation through the mouth so as to reach the alveolar sacs. Suitableformulations include aqueous or oily solutions of the active ingredient.Formulations suitable for aerosol or dry powder administration may beprepared according to conventional methods and may be delivered withother therapeutic agents such as compounds heretofore used in thetreatment or prophylaxis disorders as described below.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

The formulations may be packaged in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example water, for injection immediatelyprior to use. Extemporaneous injection solutions and suspensions areprepared from sterile powders, granules and tablets of the kindpreviously described. Preferred unit dosage formulations are thosecontaining a daily dose or unit daily sub-dose, as herein above recited,or an appropriate fraction thereof, of the active ingredient.

The invention further provides veterinary compositions comprising atleast one active ingredient as above defined together with a veterinarycarrier therefore. Veterinary carriers are materials useful for thepurpose of administering the composition and may be solid, liquid orgaseous materials which are otherwise inert or acceptable in theveterinary art and are compatible with the active ingredient. Theseveterinary compositions may be administered parenterally, orally or byany other desired route.

Combination Therapy

The compounds of Formula I may be employed alone or in combination withadditional therapeutic agents for the treatment of a disease or disorderdescribed herein, such as inflammation or a hyperproliferative disorder(e.g., cancer). In certain embodiments, a compound of Formula I iscombined in a pharmaceutical combination formulation, or dosing regimenas combination therapy, with an additional, second therapeutic compoundthat has anti-inflammatory or anti-hyperproliferative properties or thatis useful for treating an inflammation, immune-response disorder, orhyperproliferative disorder (e.g., cancer). The additional therapeuticmay be a Bcl-2 inhibitor, a Btk inhibitor, a JAK inhibitor, an anti-CD20antibody, an anti-inflammatory agent, an immunomodulatory agent,chemotherapeutic agent, an apoptosis-enhancer, a neurotropic factor, anagent for treating cardiovascular disease, an agent for treating liverdisease, an anti-viral agent, an agent for treating blood disorders, anagent for treating diabetes, and an agent for treating immunodeficiencydisorders. The second therapeutic agent may be an NSAIDanti-inflammatory agent. The second therapeutic agent may be achemotherapeutic agent. The second compound of the pharmaceuticalcombination formulation or dosing regimen preferably has complementaryactivities to the compound of Formula I such that they do not adverselyaffect each other. Such compounds are suitably present in combination inamounts that are effective for the purpose intended. In one embodiment,a composition of this invention comprises a compound of Formula I, or astereoisomer, tautomer, solvate, metabolite, or pharmaceuticallyacceptable salt or prodrug thereof, in combination with a therapeuticagent such as an NSAID.

The combination therapy may be administered as a simultaneous orsequential regimen. When administered sequentially, the combination maybe administered in two or more administrations. The combinedadministration includes coadministration, using separate formulations ora single pharmaceutical formulation, and consecutive administration ineither order, wherein preferably there is a time period while both (orall) active agents simultaneously exert their biological activities.

Suitable dosages for any of the above coadministered agents are thosepresently used and may be lowered due to the combined action (synergy)of the newly identified agent and other therapeutic agents ortreatments.

The combination therapy may provide “synergy” and prove “synergistic”,i.e., the effect achieved when the active ingredients used together isgreater than the sum of the effects that results from using thecompounds separately. A synergistic effect may be attained when theactive ingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined, unit dosage formulation; (2) delivered byalternation or in parallel as separate formulations; or (3) by someother regimen. When delivered in alternation therapy, a synergisticeffect may be attained when the compounds are administered or deliveredsequentially, e.g., by different injections in separate syringes,separate pills or capsules, or separate infusions. In general, duringalternation therapy, an effective dosage of each active ingredient isadministered sequentially, i.e., serially, whereas in combinationtherapy, effective dosages of two or more active ingredients areadministered together.

In a particular embodiment of therapy, a compound of Formula I, or astereoisomer, tautomer, solvate, metabolite, or pharmaceuticallyacceptable salt or prodrug thereof, may be combined with othertherapeutic, hormonal or antibody agents such as those described herein,as well as combined with surgical therapy and radiotherapy. Combinationtherapies according to the present invention thus comprise theadministration of at least one compound of Formula I, or a stereoisomer,tautomer, solvate, metabolite, or pharmaceutically acceptable salt orprodrug thereof, and the use of at least one other cancer treatmentmethod. The amounts of the compound(s) of Formula I and the otherpharmaceutically active therapeutic agent(s) and the relative timings ofadministration will be selected in order to achieve the desired combinedtherapeutic effect.

Metabolites of Compounds of Formula I

Also falling within the scope of this invention are the in vivometabolic products of Formula I described herein. Such products mayresult for example from the oxidation, reduction, hydrolysis, amidation,de-amidation, esterification, de-esterification, Michael additionreversal, enzymatic cleavage, and the like, of the administeredcompound. Accordingly, the invention includes metabolites of compoundsof Formula I, including compounds produced by a process comprisingcontacting a compound of this invention with a mammal for a period oftime sufficient to yield a metabolic product thereof.

Metabolite products typically are identified by preparing aradiolabelled (e.g., ¹⁴C or ³H) isotope of a compound of the invention,administering it parenterally in a detectable dose (e.g., greater thanabout 0.5 mg/kg) to an animal such as rat, mouse, guinea pig, monkey, orto man, allowing sufficient time for metabolism to occur (typicallyabout 30 seconds to 30 hours) and isolating its conversion products fromthe urine, blood or other biological samples. These products are easilyisolated since they are labeled (others are isolated by the use ofantibodies capable of binding epitopes surviving in the metabolite). Themetabolite structures are determined in conventional fashion, e.g., byMS, LC/MS or NMR analysis. In general, analysis of metabolites is donein the same way as conventional drug metabolism studies well known tothose skilled in the art. The metabolite products, so long as they arenot otherwise found in vivo, are useful in diagnostic assays fortherapeutic dosing of the compounds of the invention.

Articles of Manufacture

In another embodiment of the invention, an article of manufacture, or“kit”, containing materials useful for the treatment of the diseases anddisorders described above is provided. In one embodiment, the kitcomprises a container comprising a compound of Formula I, or astereoisomer, tautomer, solvate, metabolite, or pharmaceuticallyacceptable salt or prodrug thereof. The kit may further comprise a labelor package insert on or associated with the container. The term “packageinsert” is used to refer to instructions customarily included incommercial packages of therapeutic products, that contain informationabout the indications, usage, dosage, administration, contraindicationsand/or warnings concerning the use of such therapeutic products.Suitable containers include, for example, bottles, vials, syringes,blister pack, etc. The container may be formed from a variety ofmaterials such as glass or plastic. The container may hold a compound ofFormula I or a formulation thereof which is effective for treating thecondition and may have a sterile access port (for example, the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is a compound of Formula I. The label or package insertindicates that the composition is used for treating the condition ofchoice, such as cancer. In addition, the label or package insert mayindicate that the patient to be treated is one having a disorder such asa hyperproliferative disorder, neurodegeneration, cardiac hypertrophy,pain, migraine or a neurotraumatic disease or event. In one embodiment,the label or package inserts indicates that the composition comprising acompound of Formula I can be used to treat a disorder resulting fromabnormal cell growth. The label or package insert may also indicate thatthe composition can be used to treat other disorders. Alternatively, oradditionally, the article of manufacture may further comprise a secondcontainer comprising a pharmaceutically acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

The kit may further comprise directions for the administration of thecompound of Formula I and, if present, the second pharmaceuticalformulation. For example, if the kit comprises a first compositioncomprising a compound of Formula I and a second pharmaceuticalformulation, the kit may further comprise directions for thesimultaneous, sequential or separate administration of the first andsecond pharmaceutical compositions to a patient in need thereof.

In another embodiment, the kits are suitable for the delivery of solidoral forms of a compound of Formula I, such as tablets or capsules. Sucha kit preferably includes a number of unit dosages. Such kits caninclude a card having the dosages oriented in the order of theirintended use. An example of such a kit is a “blister pack”. Blisterpacks are well known in the packaging industry and are widely used forpackaging pharmaceutical unit dosage forms. If desired, a memory aid canbe provided, for example in the form of numbers, letters, or othermarkings or with a calendar insert, designating the days in thetreatment schedule in which the dosages can be administered.

According to one embodiment, a kit may comprise (a) a first containerwith a compound of Formula I contained therein; and optionally (b) asecond container with a second pharmaceutical formulation containedtherein, wherein the second pharmaceutical formulation comprises asecond compound with anti-hyperproliferative activity. Alternatively, oradditionally, the kit may further comprise a third container comprisinga pharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

In certain other embodiments wherein the kit comprises a composition ofFormula I and a second therapeutic agent, the kit may comprise acontainer for containing the separate compositions such as a dividedbottle or a divided foil packet, however, the separate compositions mayalso be contained within a single, undivided container. Typically, thekit comprises directions for the administration of the separatecomponents. The kit form is particularly advantageous when the separatecomponents are preferably administered in different dosage forms (e.g.,oral and parenteral), are administered at different dosage intervals, orwhen titration of the individual components of the combination isdesired by the prescribing physician.

Preparation of Formula I Compounds

Compounds of Formula I may be synthesized by synthetic routes thatinclude processes analogous to those well-known in the chemical arts,particularly in light of the description contained herein, and those forother heterocycles described in: Comprehensive Heterocyclic ChemistryII, Editors Katritzky and Rees, Elsevier, 1997, e.g. Volume 3; LiebigsAnnalen der Chemie, (9):1910-16, (1985); Helvetica Chimica Acta,41:1052-60, (1958); Arzneimittel-Forschung, 40(12):1328-31, (1990), eachof which are expressly incorporated by reference. Starting materials aregenerally available from commercial sources such as Aldrich Chemicals(Milwaukee, Wis.) or are readily prepared using methods well known tothose skilled in the art (e.g., prepared by methods generally describedin Louis F. Fieser and Mary Fieser, Reagents for Organic Synthesis, v.1-23, Wiley, N.Y. (1967-2006 ed.), or Beilsteins Handbuch derorganischen Chemie, 4, Aufl. ed. Springer-Verlag, Berlin, includingsupplements (also available via the Beilstein online database).

Synthetic chemistry transformations and protecting group methodologies(protection and deprotection) useful in synthesizing Formula I compoundsand necessary reagents and intermediates are known in the art andinclude, for example, those described in R. Larock, ComprehensiveOrganic Transformations, VCH Publishers (1989); T. W. Greene and P. G.M. Wuts, Protective Groups in Organic Synthesis, 3^(rd) Ed., John Wileyand Sons (1999); and L. Paquette, ed., Encyclopedia of Reagents forOrganic Synthesis, John Wiley and Sons (1995) and subsequent editionsthereof.

Compounds of Formula I may be prepared singly or as compound librariescomprising at least 2, for example 5 to 1,000 compounds, or 10 to 100compounds. Libraries of compounds of Formula I may be prepared by acombinatorial ‘split and mix’ approach or by multiple parallel synthesesusing either solution phase or solid phase chemistry, by proceduresknown to those skilled in the art. Thus according to a further aspect ofthe invention there is provided a compound library comprising at least 2compounds, or pharmaceutically acceptable salts thereof.

The Examples provide exemplary methods for preparing Formula Icompounds. Those skilled in the art will appreciate that other syntheticroutes may be used to synthesize the Formula I compounds. Althoughspecific starting materials and reagents are depicted and discussed inthe Figures and Examples, other starting materials and reagents can beeasily substituted to provide a variety of derivatives and/or reactionconditions. In addition, many of the exemplary compounds prepared by thedescribed methods can be further modified in light of this disclosureusing conventional chemistry well known to those skilled in the art.

In preparing compounds of Formulas I, protection of remote functionality(e.g., primary or secondary amine) of intermediates may be necessary.The need for such protection will vary depending on the nature of theremote functionality and the conditions of the preparation methods.Suitable amino-protecting groups include acetyl, trifluoroacetyl,t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz) and9-fluorenylmethyleneoxycarbonyl (Fmoc). The need for such protection isreadily determined by one skilled in the art. For a general descriptionof protecting groups and their use, see T. W. Greene, Protective Groupsin Organic Synthesis, John Wiley & Sons, New York, 1991.

Unless otherwise indicated, conventional methods of mass spectroscopy,NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniquesand pharmacology, within the skill of the art are employed. Unlessspecific definitions are provided, the nomenclature employed inconnection with, and the laboratory procedures and techniques of,analytical chemistry, synthetic organic chemistry, and medicinal andpharmaceutical chemistry described herein are those known in the art.Standard techniques can be used for chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients. Reactions and purification techniques can beperformed e.g., using kits of manufacturer's specifications or ascommonly accomplished in the art or as described herein. The foregoingtechniques and procedures can be generally performed of conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification.

Experimental procedures, intermediates and reagents useful for usefulfor the preparation of Formula I compounds may be found inWO2011/140488; US 2012/0010191; WO2013/067274; US 2013/0116235;WO2013/067277; US 2013/0116245; WO2013/067260; US 2013/0116262;WO2013/067264; US 2013/0116246, which are incorporated by reference inits entirety.

General Preparative Procedures

The Suzuki-type coupling reaction is useful to form carbon-carbon bondsto attach the rings of Formula I compounds and intermediates such as A-3(Suzuki (1991) Pure Appl. Chem. 63:419-422; Miyaura and Suzuki (1979)Chem. Reviews 95(7):2457-2483; Suzuki (1999) J. Organometal. Chem.576:147-168). Suzuki coupling is a palladium mediated cross couplingreaction of a heteroarylhalide, such as B-2 or B-4, with a boronateester such as A-1 or A-2. For example, B-2 may be combined with about1.5 equivalents of4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane), anddissolved in about 3 equivalents of sodium carbonate as a 1 molarsolution in water and an equal volume of acetonitrile. A catalyticamount, or more, of a low valent palladium reagent, such asbis(triphenylphosphine)palladium(II) dichloride, is added. In some casespotassium acetate is used in place of sodium carbonate to adjust the pHof the aqueous layer. The reaction is then heated to about 140-150° C.under pressure in a microwave reactor (Biotage AB, Uppsala, Sweden) for10 to 30 minutes. The contents are extracted with ethyl acetate, oranother organic solvent. After evaporation of the organic layer theboron ester A-1 may be purified on silica or by reverse phase HPLC.Substituents are as defined, or protected forms or precursors thereof.Likewise, bromide intermediate B-4 can be boronylated to give A-2.

Suzuki coupling of B-2 and A-2, or of A-1 and B-4, gives Formula Icompound or intermediate A-3. Boronic ester (or acid) (1.5 eq) A-1 orA-2, and a palladium catalyst such asbis(triphenylphosphine)palladium(II) chloride (0.05 eq) is added to amixture of halo intermediate (1 eq) B-2 or B-4 in acetonitrile and 1 Mof sodium carbonate aqueous solution (equal volume as acetonitrile). Thereaction mixture is heated to about 150° C. in a microwave for about 15min. LC/MS indicates whether the reaction is complete or requiresfurther time or reagents. Water is added to the mixture, and theprecipitated product is filtered and purified by HPLC to yield theproduct A-3. Substituents may be as defined, or protected forms orprecursors thereof. R⁵ is a group such as alkyl or aryl, useful forintermediates for the preparation of Formula I compounds.

A variety of low valent, Pd(II) and Pd(0) palladium catalysts,precatalysts, and ligands can be used during the Suzuki orSuzuki/Miyaura coupling step (Miyaura, N. (2002) Top. Curr. Chem.,219:11-59; Kotha, S. et al (2002) Tetrahedron, 58:9633-9695; Bellina, F.et al (2004) Synthesis, 15:2419-2440; Hassan, J. et al (2002) Chem. Rev.102:1359-1470; Littke, A. F. et al (2002) Angew. Chem., Int. Ed.41:4176-4211; Barder, T. E. et al (2005) J. Am. Chem. Soc.,127:4685-4696; Walker, S. D. et al (2004) Angew. Chem., Int. Ed.,43:1871-1876; Yin, J. et al (2002) J. Am. Chem. Soc., 124:1162-1163),including PdCl2{PtBu₂(p-R-Ph)}₂ (Guram et al (2006) Organic Letters8(9):1787-1789), PdCl₂(PPh₃)₂, Pd(t-Bu)₃, PdCl₂ dppf CH₂Cl₂, Pd(PPh₃)₄,Pd(OAc)₂/PPh₃, Cl₂Pd[(Pet₃)]₂, Pd(DIPHOS)₂, Cl₂Pd(Bipy),[PdCl(Ph₂PCH₂PPh₂)]₂, Cl₂Pd[P(o-tol)₃]₂, Pd₂(dba)₃/P(o-tol)₃,Pd₂(dba)/P(furyl)₃, Cl₂Pd[P(furyl)₃]₂, Cl₂Pd(PMePh₂)₂,Cl₂Pd[P(4-F-Ph)₃]₂, Cl₂Pd[P(C₆F₆)₃]₂, Cl₂Pd[P(2-COOH- Ph)(Ph)₂]₂,Cl₂Pd[P(4-COOH-Ph)(Ph)₂]₂, and encapsulated catalysts Pd EnCat™ 30, PdEnCat™ TPP30, and Pd(II)EnCat™ BINAP30 (US 2004/0254066).

Exemplary embodiments of low valent, Pd(II) and Pd(0) palladiumcatalysts, precatalysts, and ligands are “Buchwald” catalysts,palladacycles, and ligands, including2-Dicyclohexylphosphino-2,4,6-triisopropylbiphenyl (X-Phos, CAS Reg. No.564483-18-7) andChloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II)(X-Phos aminobiphenyl palladium chloride precatalyst, CAS Reg. No.1310584-14-5), commercially available (Johnson Matthey, West Deptford,N.J.; Sigma-Aldrich Fine Chemicals, and other suppliers). See U.S. Pat.No. 7,223,879, U.S. Pat. No. 6,395,916, U.S. Pat. No. 6,307,087.

The reaction of heteroaryl halide such as B-2, or arylhalide, such asB-4, and a boronic acid or boronic ester, to form compounds A-1 and A-2,respectively can be conducted under Buchwald palladium catalysisconditions with the Buchwald pre-catalyst palladacycle and ligandreagents in Table 3 and as described in: Biscoe et al (2008) J. Am.Chem. Soc. 130:6686-6687; Kinzel et al (2010) J. Am. Chem. Soc.132:14073-14075; Molander et al (2012) J. Am. Chem. Soc.134:11667-11673; Walker et al (2004) Angew. Chem. Int. Ed. 43:1871;Billingsley et al (2007) Angew. Chem. Int. Ed. 46:5359-5363; U.S. Pat.No. 6,946,560; U.S. Pat. No. 7,026,498; U.S. Pat. No. 7,247,731; U.S.Pat. No. 7,560,582; U.S. Pat. No. 6,307,087; U.S. Pat. No. 6,395,916;U.S. Pat. No. 7,223,879; U.S. Pat. No. 7,858,784, which are incorporatedby reference. Such reagents are commercially available (Johnson MattheyInc., Wayne, Pa.; Sigma Aldrich Fine Chemical, St. Louis, Mo.; StremChemicals, Inc., Newburyport, Mass.).

TABLE 3 Buchwald Catalysts and Ligands Name CAS Reg. No.2-Dicyclohexylphosphino-2′-(N,N- DavePhos 213697-53-1dimethylamino)biphenyl 2-Dicyclohexylphosphino-2′,4′,6′- XPhos564483-18-7 triisopropylbiphenyl 2-Dicyclohexylphosphino- SPhos657408-07-6 2′,6′-dimethoxybiphenyl 2-Di-tert-butylphosphino- tBuXPhos564483-19-8 2′,4′,6′-triisopropylbiphenyl(2-Biphenyl)dicyclohexylphosphine CyJohnPhos 247940-06-3(2-Biphenyl)di-tert-butylphosphine JohnPhos 224311-51-7 Sodium2′-dicyclohexylphosphino- SPhos [water 1049726-96-6 2,6 dimethoxy-soluble] 1,1′-biphenyl-3-sulfonate hydrate 2-Di-tert-butylphosphino-Tetramethyl 857356-94-6 3,4,5,6-tetramethyl-2′,4′,6′- tBuXPhostriisopropyl-1,1′-biphenyl 2-Dicyclohexylphosphino-2′,6′- RuPhos787618-22-8 diisopropoxybiphenyl 2′-(Diphenylphosphino)-N,N′-dimethyl-PhDave-Phos 240417-00-9 (1,1′-biphenyl)-2-amine, 2-Diphenylphosphino-2′-(N,N- dimethylamino)biphenyl2′-(Di-tert-butylphosphino)-N,N- t-BuDavePhos 224311-49-3dimethylbiphenyl-2-amine 2-Dicyclohexylphosphino-2′- MePhos 251320-86-2methylbiphenyl, 2-Methyl-2′- dicyclohexylphosphinobiphenyl2-Di-tert-butylphosphino-2′- tBuMePhos 255837-19-5 methylbiphenylAu(MeCN)SbF₆ JohnPhos 866641-66-9 (2-Biphenyl)di-tert-butylphosphineJohnPhos 854045-93-5 gold(I) chloride, 2-(Di-tert- AuClbutylphosphino)biphenyl gold(I) chloride2-Dicyclohexylphosphino-2′,4′,6′- XPhos AuCl 854045-94-6triisopropylbiphenyl gold(I) chloride 2-Dicyclohexylphosphino-2′,4′,6′-XPhos AuNTf₂ 934506-10-2 triisopropylbiphenyl gold(I)bis(trifluoromethanesulfonyl)imide 2-(Dicyclohexylphosphino)3,6-BrettPhos 1070663-78-3 dimethoxy-2′,4′,6′- triisopropyl-1,1′-biphenylRuPhos Pd G1 Methyl-t-Butyl Ether Adduct Chloro(2-dicyclohexylphosphino-XPhos 1028206-56-5 2′,4′,6′-triisopropyl-1,1′-biphenyl)[2- Palladacycle(2-aminoethyl)phenyl]palladium(II) Chloro(2-dicyclohexylphosphino- SPhos2′,6′-dimethoxy-1,1′-biphenyl)[2-(2- Palladacycleaminoethylphenyl)]palladium(II)- methyl-t-butyl ether adduct t-BuXPhospalladium(II) tBuXPhos Pd 1142811-12-8 phenethylamine chloride G12-{Bis[3,5-bis(trifluoromethyl) JackiePhos 1160861-60-8phenyl]phosphino}-3,6-dimethoxy- 2′,4′,6′-triisopropyl-1,1′-biphenyl2-(Di-tert-butylphosphino)-2′,4′,6′- tBuBrettPhos 1160861-53-9triisopropyl-3,6- dimethoxy-1,1′-biphenylDicyclohexyl(2′,4′,6′-trimethoxy[1,1′- 1000171-05-0biphenyl]-2-yl)-phosphine BrettPhos Pd G1 Methyl-t-Butyl Ether AdductChloro(2-dicyclohexylphosphino- Xphos Pd G2 1310584-14-52′,4′,6′-triisopropyl- 1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) Chloro(2-dicyclohexylphosphino- SPhos Pd G21375325-64-6 2′,6′-dimethoxy- 1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) Chloro(2-dicyclohexylphosphino- RuPhos Pd G21375325-68-0 2′,6′-diisopropoxy- 1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) Chloro[(2-dicyclohexylphosphino- CPhos-Pd-G22′,6′-bis(N,N-dimethylamino)-1,1′- biphenyl)-2-(2′-amino-1,1′-biphenyl)]palladium(II) [(2-Dicyclohexylphosphino- CPhos-Pd-G32′,6′-bis(N,N-dimethylamino)-1,1′- biphenyl)-2-(2′-amino-1,1′-biphenyl)]palladium(II) methanesulfonate [(2-Di-tert-butylphosphino-tBuXPhos-Pd- 2′,4′,6′-triisopropyl-1,1′-biphenyl)-2- G3(2′-amino-1,1′-biphenyl)]palladium(II) methanesulfonate(2-Dicyclohexylphosphino- RuPhos-Pd-G32′,6′-diisopropoxy-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) methanesulfonate(2-Dicyclohexylphosphino- XPhos-Pd-G32′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) methanesulfonate[(2-Di-cyclohexylphosphino- BrettPhos-Pd-3,6-dimethoxy-2′,4′,6′-triisopropyl- G3 1,1′-biphenyl)-2-(2′-amino-1,1′-biphenyl)]palladium(II) methanesulfonate[(2-{Bis[3,5-bis(trifluoromethyl) JackiePhos-Pd-phenyl]phosphine}-3,6-dimethoxy- G32′,4′,6′-triisopropyl-1,1′-biphenyl)-2-(2′-amino-1,1′-biphenyl)]palladium(II) methanesulfonate Me4-tert-butylXPhos-AuMeCN SbF6 1334547-72-6 tBuXPhos Au(MeCN)SbF₆ 1140531-94-7 RuPhosAu(MeCN)SbF₆ SPhos Au(MeCN)SbF₆ 1236160-37-4 XPhos Au(MeCN)SbF₆1215877-64-7 Me4-tert-butyl XPhos-AuCl 1140907-91-0 tBuXPhos AuCl RuPhosAuCl 1261452-57-6 SPhos AuCl 854045-95-7 CyJohnPhos AuCl 854045-92-4BrettPhos AuCl 1334547-75-9 JohnPhos AuNTf₂ 1036000-94-8 Me₄-tert-butylXPhos-AuNTf₂ tBuXphos AuNTf₂ 1190991-33-3 SPhos AuNTf₂ 1121960-90-4CyJohnPhos AuNTf₂ 1016161-75-3 CPhos AuNTf₂ RuPhos AuNTf₂ BrettPhosAuNTf₂ 1296269-97-0 DavePhos AuNTf₂ 1188507-66-5 CPhos 1160556-64-8Chloro(sodium-2- dicyclohexylphosphino-2′,6′-dimethoxy-1,1′-biphenyl-3′- sulfonate)[2-(2′-amino-1,1′-biphenyl)]palladium(II) Di-Ad-BrettPhos 1160861-59-5Dicyclohexyl(2-(2-methoxynaphthalen-1- 1309570-98-6 yl)phenyl)phosphinetert-BuBrettPhos-Pd-G3 di-Ad-Johnphos-G3Methods of Separation

In the methods of preparing Formula I compounds, it may be advantageousto separate reaction products from one another and/or from startingmaterials. The desired products of each step or series of steps isseparated and/or purified to the desired degree of homogeneity by thetechniques common in the art. Typically such separations involvemultiphase extraction, crystallization from a solvent or solventmixture, distillation, sublimation, or chromatography. Chromatographycan involve any number of methods including, for example: reverse-phaseand normal phase; size exclusion; ion exchange; high, medium and lowpressure liquid chromatography methods and apparatus; small scaleanalytical; simulated moving bed (SMB) and preparative thin or thicklayer chromatography, as well as techniques of small scale thin layerand flash chromatography.

Another class of separation methods involves treatment of a mixture witha reagent selected to bind to or render otherwise separable a desiredproduct, unreacted starting material, reaction by product, or the like.Such reagents include adsorbents or absorbents such as activated carbon,molecular sieves, ion exchange media, or the like. Alternatively, thereagents can be acids in the case of a basic material, bases in the caseof an acidic material, binding reagents such as antibodies, bindingproteins, selective chelators such as crown ethers, liquid/liquid ionextraction reagents (LIX), or the like. Selection of appropriate methodsof separation depends on the nature of the materials involved, such as,boiling point and molecular weight in distillation and sublimation,presence or absence of polar functional groups in chromatography,stability of materials in acidic and basic media in multiphaseextraction, and the like.

Diastereomeric mixtures can be separated into their individualdiastereomers on the basis of their physical chemical differences bymethods well known to those skilled in the art, such as bychromatography and/or fractional crystallization. Enantiomers can beseparated by converting the enantiomeric mixture into a diastereomericmixture by reaction with an appropriate optically active compound (e.g.,chiral auxiliary such as a chiral alcohol or Mosher's acid chloride),separating the diastereomers and converting (e.g., hydrolyzing) theindividual diastereoisomers to the corresponding pure enantiomers. Also,some of the compounds of the present invention may be atropisomers(e.g., substituted biaryls) and are considered as part of thisinvention. Enantiomers can also be separated by use of a chiral HPLCcolumn.

A single stereoisomer, e.g., an enantiomer, substantially free of itsstereoisomer may be obtained by resolution of the racemic mixture usinga method such as formation of diastereomers using optically activeresolving agents (Eliel, E. and Wilen, S. “Stereochemistry of OrganicCompounds,” John Wiley & Sons, Inc., New York, 1994; Lochmuller, C. H.,(1975) J. Chromatogr., 113(3):283-302). Racemic mixtures of chiralcompounds of the invention can be separated and isolated by any suitablemethod, including: (1) formation of ionic, diastereomeric salts withchiral compounds and separation by fractional crystallization or othermethods, (2) formation of diastereomeric compounds with chiralderivatizing reagents, separation of the diastereomers, and conversionto the pure stereoisomers, and (3) separation of the substantially pureor enriched stereoisomers directly under chiral conditions. See: “DrugStereochemistry, Analytical Methods and Pharmacology,” Irving W. Wainer,Ed., Marcel Dekker, Inc., New York (1993).

Under method (1), diastereomeric salts can be formed by reaction ofenantiomerically pure chiral bases such as brucine, quinine, ephedrine,strychnine, α-methyl-β-phenylethylamine (amphetamine), and the like withasymmetric compounds bearing acidic functionality, such as carboxylicacid and sulfonic acid. The diastereomeric salts may be induced toseparate by fractional crystallization or ionic chromatography. Forseparation of the optical isomers of amino compounds, addition of chiralcarboxylic or sulfonic acids, such as camphorsulfonic acid, tartaricacid, mandelic acid, or lactic acid can result in formation of thediastereomeric salts.

Alternatively, by method (2), the substrate to be resolved is reactedwith one enantiomer of a chiral compound to form a diastereomeric pair(E. and Wilen, S. “Stereochemistry of Organic Compounds”, John Wiley &Sons, Inc., 1994, p. 322). Diastereomeric compounds can be formed byreacting asymmetric compounds with enantiomerically pure chiralderivatizing reagents, such as menthyl derivatives, followed byseparation of the diastereomers and hydrolysis to yield the pure orenriched enantiomer. A method of determining optical purity involvesmaking chiral esters, such as a menthyl ester, e.g., (−) menthylchloroformate in the presence of base, or Mosher ester,α-methoxy-α-(trifluoromethyl)phenyl acetate (Jacob III. J. Org. Chem.(1982) 47:4165), of the racemic mixture, and analyzing the ¹H NMRspectrum for the presence of the two atropisomeric enantiomers ordiastereomers. Stable diastereomers of atropisomeric compounds can beseparated and isolated by normal- and reverse-phase chromatographyfollowing methods for separation of atropisomeric naphthyl-isoquinolines(WO 96/15111). By method (3), a racemic mixture of two enantiomers canbe separated by chromatography using a chiral stationary phase (“ChiralLiquid Chromatography” (1989) W. J. Lough, Ed., Chapman and Hall, NewYork; Okamoto, J. Chromatogr., (1990) 513:375-378). Enriched or purifiedenantiomers can be distinguished by methods used to distinguish otherchiral molecules with asymmetric carbon atoms, such as optical rotationand circular dichroism.

EXAMPLES Example 101N-{2-[(6-{[5-(2-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-10-yl}-3-(hydroxymethyl)pyridin-4-yl)-1-methyl-2-oxo-1,2-dihydropyridin-3-yl]amino}pyridin-2-yl)oxy]ethyl}prop-2-enamide101

Step 1: 5-Bromo-3-(6-hydroxypyridin-2-ylamino)-1-methylpyridin-2(1H)-one101a

A 100 mL round-bottomed flask equipped with a reflux condenser wascharged with 6-aminopyridin-2-ol (1.1 g, 10.0 mmol),3,5-dibromo-1-methylpyridin-2(1H)-one (2.67 g, 10.0 mmol), cesiumcarbonate (6.52 g, 20.0 mmol), xantphos (576 mg, 1.0 mmol), Pd₂(dba)₃(460 mg, 0.50 mmol), and DMF (35 mL). The system was subject to threecycles of vacuum/argon flush and heated at 100° C. for 3 h. It was thencooled to room temperature and filtered. The filtrate was diluted withDCM (500 mL) and washed with H₂O (80 mL×3). The organic layer was driedand concentrated under reduced pressure. The residue solid was purifiedby silica-gel column-chromatography eluting with DCM/MeOH (30:1 to 15:1)to afford 101a (580 mg, 20%) as yellow solid. MS-ESI: [M+H]⁺ 296.0

Step 2: Tert-butyl2-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-2-yloxy)ethylcarbamate101b

To a mixture of tert-butyl2-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)-pyridin-2-yloxy)ethylcarbamate(500 mg, 1.70 mmol), tert-butyl 2-hydroxyethylcarbamate (1.1 g, 6.8mmol), and PPh₃ (1.78 g, 6.8 mmol) at 0° C. in anhydrous THF (40 mL),was added dropwise a solution of diisopropyl azodiformate (1.37 g, 6.8mmol). This mixture was heated at 40° C. for 3 h. It was then cooled toroom temperature and concentrated under reduced pressure. The residuewas partitioned between H₂O and DCM. The combined organic layer wasdried and concentrated under reduced pressure. The residue solid waspurified by silica-gel column-chromatography eluting with DCM/MeOH (60:1to 40:1) to afford 101b (520 mg, 70%) as yellow solid. MS-ESI: [M+H]⁺438.9

Step 3:[4-(5-{[6-(2-{[(Tert-butoxy)carbonyl]amino}ethoxy)pyridin-2-yl]amino}-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-10-yl}pyridin-3-yl]methylacetate 101d

A 50 mL round-bottomed flask equipped with a reflux condenser wascharged with 101b (500 mg, 1.14 mmol),[4-(dihydroxyboranyl)-2-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-10-yl}pyridin-3-yl]methylacetate 101c (452 mg, 1.14 mmol), K₃PO₄ (483 mg, 2.28 mmol), NaOAc (187mg, 2.28 mmol), Pd(dppf)Cl₂ (42 mg, 0.057 mmol), and CH₃CN/H₂O (20.0/0.5mL). The system was subject to three cycles of vacuum/argon flush thenheated at 95° C. under N₂ for 1 h. The reaction mixture was cooled toroom temperature and concentrated under reduced pressure. The residuewas purified by silica-gel column-chromatography eluting with DCM/MeOH(70:1 to 30:1) to afford 101d (400 mg, 50%) as yellow solid. MS-ESI:[M+H]⁺ 712.3

Step 4:[4-(5-{[6-(2-Aminoethoxy)pyridin-2-yl]amino}-1-methyl-6-oxo-1,6-dihydro-pyridin-3-yl)-2-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-10-yl}pyridin-3-yl]methylacetate 101e

To a solution 101d (400 mg, 0.56 mmol) in DCM (15 mL) was added dropwise3M HCl (in dioxane). This mixture was stirred at room temperature for 2h. The mixture was concentrated under reduced pressure. The residualyellow solid was washed with ethyl acetate and dried under vacuum toafford 101e (290 mg, 80%) as a yellow solid. MS-ESI: [M+H]⁺ 612.3

Step 5:(2-{4,4-Dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-10-yl}-4-[1-methyl-6-oxo-5-({6-[2-(prop-2-enamido)ethoxy]pyridin-2-yl}amino)-1,6-dihydropyridin-3-yl]pyridin-3-yl)methylacetate 101f

To a solution of 101e (250 mg, 0.387 mmol) and TEA (78 mg, 0.77 mmol) inDCM (10 mL) was added dropwise a solution of acryloyl chloride (42 mg,0.46 mmol) in DCM. This mixture was stirred at room temperature for 2 h.The mixture was concentrated under reduce pressure to afford 101f as ayellow solid (crude), which was used in next step without furtherpurification. MS-ESI: [M+H]⁺ 666.4

—Step 6: To a solution of 101f (257 mg, 0.387 mmol) in THF/i-PrOH/H₂O(5.0/3.0/2.0 mL) was added LiOH (46 mg, 1.95 mmol) at room temperature.The reaction was stirred for 1 h. The mixture was diluted with water (15mL) and extracted with DCM (15 mL×3). The combined organic layer waswashed with brine (10 mL), dried over Na₂SO₄, filtered, and concentratedunder reduced pressure. The residue solid was purified by reverse-phaseprep-HPLC to afford 101 (95 mg, 39% two steps) as a white solid. MS-ESI:[M+H]⁺ 624.2. ¹H NMR (500 MHz, CDCl₃) δ 8.74 (d, J=2.0 Hz, 1H), 8.53 (d,J=5.0 Hz, 1H), 9.70 (s, 1H), 7.73 (d, J=2.0 Hz, 1H), 7.50-7.48 (m, 1H),7.29-7.27 (m, 1H), 6.85 (s, 1H), 6.46-6.44 (m, 2H), 6.27-6.23 (m, 2H),6.05-6.00 (m, 1H), 5.61-5.59 (m, 1H), 4.98-4.96 (m, 1H), 4.72-4.70 (m,1H), 4.55-4.53 (m, 1H), 4.39-4.29 (m, 3H), 4.19-4.16 (m, 2H), 3.96-3.94(m, 1H), 375-3.73 (m, 4H, overlap), 3.64-3.60 (m, 1H), 2.61-2.59 (m,2H), 2.53-2.51 (m, 2H), 1.30 (s, 6H).

Example 102N-(cyanomethyl)-1-(4-{[5-(2-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-10-yl}-3-(hydroxymethyl)pyridin-4-yl)-1-methyl-2-oxo-1,2-dihydropyridin-3-yl]amino}pyrimidin-2-yl)pyrrolidine-3-carboxamide102

Step 1: 1-(4-Aminopyrimidin-2-yl) pyrrolidine-3-carboxylic acid 102a

A 100 mL round-bottomed flask equipped with a magnetic stirrer and areflux condenser was charged with pyrrolidine-3-carboxylic acid (1.20 g,10 mmol), 2-chloropyrimidin-4-amine (1.30 g, 10 mmol), isopropyl alcohol(IPA, 40 mL), and TEA (6 mL). The mixture was heated at 80° C. overnight(O/N). After this time the reaction was cooled to room temperature(r.t.). It was then filtered and the filter cake was washed with DCM toafford 102a (1.4 g, 67%) as a pale yellow solid. MS: [M+H]⁺ 209.1

Step 2:1-(4-{[5-(2-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-10-yl}-3-(hydroxymethyl)pyridin-4-yl)-1-methyl-2-oxo-1,2-dihydropyridin-3-yl]amino}pyrimidin-2-yl)pyrrolidine-3-carboxylicacid 102c.

A 100 mL round-bottomed flask equipped with a magnetic stirrer and areflux condenser was charged with10-[4-(5-bromo-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-3-(hydroxymethyl)pyridin-2-yl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-9-one102b (497 mg, 1.0 mmol), 102a (312 mg, 1.5 mmol), Pd₂(dba)₃ (92 mg, 0.10mmol), XantPhos (4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene, CASReg. No. 161265-03-8, 116 mg, 0.20 mmol), Cs₂CO₃ (652 mg, 2.0 mmol), andDMF (20 mL). After three cycles of vacuum/argon flush, the mixture washeated at 80° C. for 4 h. After this time the reaction was cooled toroom temperature. It was then filtered and the filtrate was evaporatedunder reduced pressure. The residue was purified by reverse-phaseprep-HPLC to afford 102c (82 mg, 13%) as a white solid. MS: [M+H]⁺ 625.4

Step 3: A 50 mL round-bottomed flask equipped with a magnetic stirrerand a reflux condenser was charged with 102c (100 mg, 0.16 mmol),N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (HATU, 152 mg, 0.40 mmol), triethylamine (TEA, 8drops), THF (10 mL), and 2-aminoacetonitrile (5 drops). The mixture washeated at 30° C. for 5 h. After this time the reaction was cooled toroom temperature. It was then filtered and the filtrate was evaporatedunder reduced pressure. The residue was purified by reverse-phaseprep-HPLC to afford 102 (30 mg, 28%) as a white solid. MS: [M+H]⁺ 663.2.¹H NMR (500 MHz, DMSO-d₆) δ 8.88 (d, J=6.0 Hz, 1H), 8.81 (s, 1H), 8.74(s, 1H), 8.47 (d, J=5.0 Hz, 1H), 7.95 (d, J=5.5 Hz, 1H), 7.71 (s, 1H),7.35 (d, J=4.5 Hz, 1H), 6.58-6.55 (m, 2H), 5.02 (s, 1H), 4.53-4.51 (m,1H), 4.43-4.40 (m, 1H), 4.23-4.15 (m, overlap, 5H), 3.86-3.84 (m, 1H),3.72-3.69 (m, 1H), 3.62 (s, 3H), 3.60-3.56 (m, 1H), 3.51-3.43 (m, 2H),3.10-3.08 (m, 1H), 2.62-2.54 (m, 2H), 2.43 (s, 2H), 2.18-2.16 (m, 1H),2.06-2.04 (m, 1H), 1.22 (s, 6H).

Example 103N-[2-[[6-[[5-[5-fluoro-2-(hydroxymethyl)-3-(4-oxo-6,7,8,9-tetrahydrobenzothiopheno[2,3-d]pyridazin-3-yl)phenyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]oxy]ethyl]prop-2-enamide103

Step 1:3-(6-(2-aminoethoxyl)pyridin-2-ylamino)-5-bromo-1-methylpyridin-2(1H)-onehydrochloride 103a

To a mixture of tert-butyl 2-hydroxyethylcarbamate (6.4 g, 40 mmol) andEt₃N (7.2 mL, 52 mmol) in DCM (50 mL) was added para-toluenesulfonylchloride, TsCl (8.4 g, 44 mmol). The mixture was stirred at roomtemperature (rt) overnight (ON). The reaction mixture was concentratedand the residue was partitioned between EA and water. The organic layerwas separated, dried over Na₂SO₄, filtered and concentrated. The residuewas purified by silica gel chromatography (PE/EA=5/1) to give2-(tert-butoxycarbonylamino)ethyl 4-methylbenzenesulfonate (10.0 g, 79%)as white solid.

A mixture of 3,5-dibromo-1-methylpyridin-2(1H)-one (2.67 g, 10.0 mmol),6-aminopyridin-2-ol (1.1 g, 10.0 mmol), Pd₂(dba)₃ (460 mg, 0.5 mmol),Xantphos (576 mg, 1.0 mmol) and Cs₂CO₃ (6.52 g, 20.0 mmol) in DMF (40mL) was stirred at 100° C. overnight. The mixture was concentrated andthe residue was treated with DCM. The precipitate was collected byfiltration and dried to give5-bromo-3-(6-hydroxypyridin-2-ylamino)-1-methylpyridin-2(1H)-one 101a(4.0 g crude) as a brown solid.

A mixture of compound 101a (7.0 g, 23.75 mmol),2-(tert-butoxycarbonylamino)ethyl 4-methylbenzenesulfonate (3.0 g, 9.5mmol) and Cs₂CO₃ (3.7 g, 11.4 mmol) in DMF (40 mL) was stirred at 100°C. for 4 h. The reaction mixture was concentrated. The residue wastreated with EA and filtered. The filtrate was washed with water. Theorganic layer was separated, dried over Na₂SO₄, filtered andconcentrated to give Tert-butyl2-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-2-yloxy)ethylcarbamate101b (650 mg, 15% for two steps) as a yellow solid. A mixture of 101b(700 mg, 1.6 mmol) in HCl (5 mL, 4M in 1,4-dioxane, 20 mmol) was stirredat rt overnight. The reaction mixture was concentrated to give 103a (550mg, 83%) as yellow solid. MS-ESI: [M+H]⁺ 341.0

Step 2:3-(6-(2-aminoethoxyl)pyridin-2-ylamino)-5-bromo-1-methylpyridin-2(1H)-onehydrochloride 103b

A mixture of 103a (900 mg, 2.4 mmol) and Et₃N (0.83 mL, 6.0 mmol) in DCM(15 mL) was added acryloyl chloride (0.23 mL, 2.88 mmol). The mixturewas stirred at rt for 2 h and quenched with water. The organic layer wasseparated, dried over Na₂SO₄, filtered and concentrated to give 103b(620 mg, 66%) as light yellow solid. MS-ESI: [M+H]⁺ 393.1

Step 3:N-(2-(6-(1-methyl-2-oxo-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2-dihydropyridin-3-ylamino)pyridin-2-yloxy)ethyl)acrylamide103c

A mixture of 103b (250 mg, 0.64 mmol),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane), Pin₂B₂ (570mg, 2.23 mmol), Pd₂(dba)₃ (116 mg, 0.128 mmol), Xphos (122 mg, 0.256mmol) and KOAc (125 mg, 1.28 mmol) in dioxane (16 mL) was stirred at 65°C. for 4 h under N₂. The reaction mixture was concentrated to give 103c(825 mg crude) as brown solid. This material was used for the next stepdirectly. MS-ESI: [M+H]⁺ 441.2

Step 4: A mixture of 103c (250 mg, 0.57 mmol),6-(3-bromo-5-fluoro-2-(hydroxymethyl)phenyl)-6,7,8,9-tetrahydrodibenzo[b,d]thieno[3,2-d]pyridazin-7(6H)-one103d (193 mg, 0.47 mmol), Pd(dppf)Cl₂ (71 mg, 0.094 mmol) and K₃PO₄ (250mg, 1.18 mmol) in acetonitrile (10 mL) and water (1 mL) was stirred at90° C. for 5 h under N₂. The mixture was filtered and the filtrate wasconcentrated. The residue was purified by prep-HPLC to give 103 (40 mg,10% for two steps) as a white solid. ¹H NMR (500 MHz, CDCl₃): δ 8.64 (s,1H), 8.27 (s, 1H), 7.93 (s, 1H), 7.52-7.49 (m, 1H), 7.35 (s, 1H),7.27-7.25 (m, 1H), 7.14 (m, 1H), 6.61 (s, 1H), 6.47 (m, 1H), 6.23-6.22(m, 1H), 5.98-5.92 (m, 1H), 5.51 (d, J=10.0 Hz, 1H), 4.40 (s, 2H),4.29-4.27 (m, 2H), 3.74 (s, 3H), 3.52 (s, 2H), 3.00-2.98 (m, 2H),2.88-2.87 (m, 2H), 1.99-1.97 (m, 4H). MS-ESI: [M+H]⁺ 643.2

Example 104N-[2-[[6-[[5-[2-(6-tert-butyl-8-fluoro-1-oxo-phthalazin-2-yl)-3-(hydroxymethyl)-4-pyridyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]oxy]ethyl]prop-2-enamide104

Step 1:(2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)-4-chloropyridin-3-yl)methylacetate 104b

To a mixture of6-tert-butyl-2-(4-chloro-3-(hydroxymethyl)pyridin-2-yl)-8-fluorophthalazin-1(2H)-one104a (150 mg, 0.42 mmol) and triethylamine, Et₃N (0.12 mL, 0.84 mmol) intetrahydrofuran, THF (2.5 mL) was added acetyl chloride, AcCl (44 μl,0.62 mmol). The mixture was stirred at rt for 1 h and quenched withwater, extracted with ethylacetate, EA. The combined organic layers weredried over Na₂SO₄, filtered and concentrated to give 104b (160 mg) as acolorless oil. MS-ESI: [M+H]⁺ 404.1

Step 2:3-(acetoxymethyl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)pyridin-4-ylboronicacid 104c

A mixture of 104b (150 mg, 0.37 mmol),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane), Pin₂B₂ (379mg, 1.48 mmol), Pd(dppf)Cl₂ (28 mg, 0.037 mmol), Xphos (35 mg, 0.074mmol) and KOAc (108 mg, 1.11 mmol) in dioxane (5 mL) was stirred at 65°C. for 15 h under N₂. The reaction mixture was concentrated. The residuewas washed with DCM/PE (1 mL/20 mL) and filtered. The filtrate wasconcentrated to give 104c (100 mg, 66%) as yellow solid. MS-ESI: [M+H]⁺414.2

Step 3:(4-(5-(6-(2-acrylamidoethoxyl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(6-tert-butyl-8-fluoro-1-oxophthalazin-2(1H)-yl)pyridin-3-yl)methylacetate 104d

A mixture of 104c (256 mg, 0.62 mmol),3-(6-(2-aminoethoxyl)pyridin-2-ylamino)-5-bromo-1-methylpyridin-2(1H)-onehydrochloride 103b (120 mg, 0.31 mmol), Pd(dppf)Cl₂ (47 mg, 0.062 mmol)and K₃PO₄ (131 mg, 0.62 mmol) in acetonitrile (2 mL) and water (0.2 mL)was stirred at 90° C. for 3 h under N₂. The mixture was filtered and thefiltrate was concentrated. The residue was washed with petroleum ether,PE to give 104d (160 mg) as brown solid. This material was used for thenext step directly.

Step 4: A mixture of 104d (140 mg, 0.21 mmol) and LiOH (44 mg, 1.05mmol) in THF/i-PrOH/water (2.0 mL/1.2 mL/0.8 mL) was stirred at rt for 1h. The mixture was extracted with EA. The combined organic layers weredried over Na₂SO₄, filtered and concentrated. The residue was purifiedby prep-HPLC to give 104 (10 mg, 10% for two steps) as a light yellowsolid. ¹H NMR (500 MHz, CDCl₃): δ 8.72-8.66 (m, 2H), 8.33 (s, 1H), 7.91(s, 1H), 7.59-7.47 (m, 6H), 6.51 (s, 1H), 6.44 (d, J=10.0 Hz, 1H),6.24-6.16 (m, 2H), 5.99-5.94 (m, 1H), 5.50 (d, J=10.0 Hz, 1H), 4.58-4.48(m, 2H), 4.28-4.21 (m, 2H), 3.99-3.57 (m, 5H), 1.43 (s, 9H). MS-ESI:[M+H]⁺ 640.3

Example 105N-[2-[[6-[[5-[5-fluoro-2-(hydroxymethyl)-3-(4-oxo-6,7,8,9-tetrahydrobenzothiopheno[2,3-d]pyridazin-3-yl)phenyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]amino]ethyl]prop-2-enamide105

Step 1: A mixture of 6-bromopyridin-2-amine (17.2 g, 100 mmol), Boc₂O(45.9 mL, 200 mmol), Et₃N (40.4 mL, 300 mmol), DMAP (610 mg, 5.0 mmol)and t-BuOH (200 mL) was stirred at 60° C. for 16 h. The reaction mixturewas cooled to 0° C., filtered and dried to give N,N-bis-Boc6-bromopyridin-2-amine 105a (24.0 g, 65%) as white solid. ¹H NMR (500MHz, CDCl3): δ 7.58-7.25 (m, 3H), 1.46 (s, 18H).

Step 2: A mixture of 105a (3.72 g, 10 mmol), tert-butyl2-aminoethylcarbamate (1.60 g, 10 mmol), Pd₂(dba)₃ (91.6 mg, 0.1 mmol)Xantphos (116 mg, 0.2 mmol), Cs₂CO₃(9.78 g, 30 mmol) in dioxane (100 mL)was degassed and stirred at 95° C. for 6 h under nitrogen. The reactionmixture was concentrated and the residue was purified by silica gelchromatography (EA/PE=1/1) to give N,N-bis-Boc6-(2-N-Boc-aminoethyl)pyridin-2-amine 105b (2.62 g, 58%) as yellowsolid. MS-ESI: [M+H]⁺ 453.3

Step 3: To a mixture of 105b (4.52 g, 10 mmol) in dioxane (50 mL) wasadded HCl (4M in dioxane, 10 mL, 40 mmol). The reaction mixture wasstirred at rt for 16 h and concentrated to giveN2-(2-aminoethyl)pyridine-2,6-diamine hydrochloride 105c (1.27 g, 84%)as yellow solid.

Step 4: A mixture of 105c (1.88 g, 10 mmol), acrylic acid (720 mg, 10mmol), HATU (3.80 g, 10 mmol) and DIPEA (6.97 mL, 40 mmol) in DCM (50mL) was stirred at rt for 16 h. The reaction mixture was concentratedand the residue was purified by silica gel chromatography (EA/MeOH=4/1)to give N-(2-(6-aminopyridin-2-ylamino)ethyl)acrylamide 105d (1.07 g,52%) as pale-yellow oil.

Step 5: A mixture of 105d (2.06 g, 10 mmol),3,5-dibromo-1-methylpyridin-2(1H)-one (2.65 g, 10 mmol), Pd₂(dba)₃ (91.6mg, 0.1 mmol) Xant-phos (116 mg, 0.2 mmol), Cs₂CO₃(9.78 g, 30 mmol) indioxane (100 mL) was degassed and stirred at 100° C. for 16 h undernitrogen. The reaction mixture was concentrated and the residue waspurified by silica gel chromatography (EA/PE=5/1) to giveN-(2-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-2-ylamino)ethyl)acrylamide105e (2.54 g, 65%) as yellow solid. MS-ESI: [M+H]⁺ 394.1.

Step 6: A mixture of 105e (235 mg, 0.601 mmol),(4-fluoro-2-{6-oxo-8-thia-4,5-diazatricyclo[7.4.0.02,7]trideca-1(9),2(7),3-triene-5-yl}-6-(tetra-methyl-1,3,2-dioxaborolan-2-yl)phenyl)methylacetate 108c (299 mg, 0.601 mmol), Pd(dppf)Cl₂(42.8 mg, 0.06 mmol) andK₃PO₄(382 mg, 1.80 mmol) in ACN (10 mL) and H₂O (2 mL) was degassed andstirred at 85° C. for 4 h under nitrogen. The reaction mixture wasconcentrated and the residue was purified by silica gel chromatography(EA/PE=4/1) to giveN-[2-[[6-[[5-[5-fluoro-2-(acetoxymethyl)-3-(4-oxo-6,7,8,9-tetrahydrobenzothiopheno[2,3-d]pyridazin-3-yl)phenyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]amino]ethyl]prop-2-enamide105f (125 mg, 31%) as a brown solid. MS-ESI: [M+H]⁺ 684.3

Step 7: To a mixture of 105f (125 mg, 0.183 mmol) in THF (6 mL), i-PrOH(2 mL) and H₂O (2 mL) was added LiOH.H₂O (37 mg, 0.915 mmol). Thereaction mixture was stirred at rt for 16 h and concentrated. Theresidue was partitioned between water and DCM. The organic layer wasseparated, dried over Na₂SO₄, filtered and concentrated. The residue waswashed with EA and dried to give 105 (73 mg, 60%) as gray solid. ¹H NMR(500 MHz, DMSO-d₆): δ 8.56 (s, 1H), 8.48 (s, 1H),), 8.20 (s, 1H), 8.12(s, 1H), 7.42 (s, 1H), 7.38-7.32 (m, 2H), 7.23 (t, J=8.0 Hz, 1H), 6.52(s, 1H), 6.35 (d, J=8.0 Hz, 1H), 6.21-6.15 (m, 1H), 6.05-6.02 (m, 1H),5.95 (d, J=8.0 Hz, 1H), 4.73 (s, 1H), 4.33 (s, 2H), 3.59 (s, 3H),3.28-3.25 (m, 5H), 2.93 (bs, 2H), 2.84 (bs, 2H), 1.88 (bs, 4H).ESI-LCMS: m/z=642 [C34H34FN7O5+H]+

Example 106N-[2-[[6-[[5-[5-fluoro-2-(hydroxymethyl)-3-(4-oxo-6,7,8,9-tetrahydrobenzothiopheno[2,3-d]pyridazin-3-yl)phenyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]oxy]ethyl]but-2-ynamide106

Step 1: A mixture ofN-(2-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-2-yloxy)ethyl)but-2-ynamide116a (224 mg, 0.45 mmol),(4-fluoro-2-{6-oxo-8-thia-4,5-diazatricyclo[7.4.0.02,7]trideca-1(9),2(7),3-triene-5-yl}-6-(tetra-methyl-1,3,2-dioxaborolan-2-yl)phenyl)methylacetate 108c (240 mg, 0.59 mmol), Pd(dppf)Cl₂ (90 mg, 0.12 mmol) andK₃PO₄ (250 mg, 1.18 mmol) in acetonitrile (10 mL) and water (1 mL) wasstirred at 90° C. for 3 h under N₂. The mixture was filtered and thefiltrate was concentrated to giveN-[2-[[6-[[5-[5-fluoro-2-(acetoxymethyl)-3-(4-oxo-6,7,8,9-tetrahydrobenzothiopheno[2,3-d]pyridazin-3-yl)phenyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]oxy]ethyl]but-2-ynamide106a (330 mg crude) as a brown solid. ESI-LCMS: m/z=696.8. This materialwas used for the next step directly

Step 2: A mixture of 106a (330 mg, 0.47 mmol) and LiOH.H₂O (196 mg, 4.67mmol) in THF/i-PrOH/water (5.0 mL/3.0 mL/2.0 mL) was stirred at rt for 1h. The mixture was extracted with EA. The combined organic layers weredried over Na₂SO₄, filtered and concentrated. The residue was purifiedby prep-HPLC to give 106 (30 mg, 8% for two steps) as white solid. ¹HNMR (500 MHz, CDCl₃): δ 8.52 (s, 1H), 8.26 (s, 1H), 7.52 (t, J=8.0 Hz,1H), 7.45 (s, 1H), 7.13-7.10 (m, 1H), 6.69 (s, 1H), 6.52 (d, J=8.0 Hz,1H), 6.22 (d, J=8.5 Hz, 1H), 4.34 (s, 2H), 4.27 (t, J=5.0 Hz, 2H),2.98-2.97 (m, 2H), 2.87-2.85 (m, 2H), 1.99-1.96 (m, 4H), 1.86 (s, 3H).ESI-LCMS: m/z=655 [C34H31FN6O5S+H]+

Example 107N-[(1S)-2-[[6-[[5-[2-(7,7-dimethyl-4-oxo-1,2,6,8-tetrahydrocyclopenta[3,4]pyrrolo[3,5-b]pyrazin-3-yl)-3-(hydroxymethyl)-4-pyridyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]oxy]-1-methyl-ethyl]prop-2-enamide107

Step 1: (S)-2-(tert-butoxycarbonylamino)propyl methanesulfonate 107a

To an ice-cooled mixture of (S)-tert-butyl 1-hydroxypropan-2-ylcarbamate(3.0 g, 17.1 mmol) and Et₃N (4.8 mL, 34.2 mmol) in DCM (50 mL) was addedmethanesulfonyl chloride, MsCl (1.33 mL, 17.1 mmol) dropwise. Thereaction mixture was stirred at rt for 2 h, diluted with DCM, washedwith water. The organic layer was separated, dried over Na₂SO₄, filteredand concentrated to give 107a (3.72 g, 86%) as a white solid. ¹H NMR(500 MHz, DMSO-d6): δ 4.044-4.033 (d, 2H), 3.164 (s, 3H), 1.385 (s, 9H),1.065-1.051 (d, 3H)

Step 2: (S)-tert-butyl1-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-2-yloxy)propan-2-ylcarbamate107b

A mixture of 107a (964 mg, 5.5 mmol),5-Bromo-3-(6-hydroxypyridin-2-ylamino)-1-methylpyridin-2(1H)-one 101a (4g crude, 16.5 mmol), Cs₂CO₃ (5.38 g, 11 mmol) in DMF (20 mL) was stirredat 95° C. for 16 h. The reaction mixture was diluted with DCM, washedwith water. The organic layer was separated, dried over Na₂SO₄, filteredand concentrated. The residue was purified by silica gel chromatography(DCM/MeOH=20/1) to give 107b (550 mg, 22%) as a brown solid. MS-ESI:[M+H]⁺ 453.1

Step 3:(S)-3-(6-(2-aminopropoxyl)pyridin-2-ylamino)-5-bromo-1-methylpyridin-2(1H)-onehydrochloride 107c

A mixture of 107b (550 mg, 1.21 mmol) in DCM (10 mL) was treated withHCl (4 M in dioxone, 3 mL, 12 mmol). The reaction mixture was stirred atrt for 2 h and quenched with sat. Na₂CO₃, extracted with DCM. Thecombined extracts were dried over Na₂SO₄, filtered and concentrated togive 107c (400 mg, 1.03 mmol) as brown solid. MS-ESI: [M+H]⁺ 352.9

Step 4:(S)—N-(1-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-2-yloxy)propan-2-yl)acrylamide107d

To an ice-cooled mixture of 107c (390 mg, 1.0 mmol) and TEA (0.53 ml,3.0 mmol) in DCM (20 mL) was added acryloyl chloride (0.23 ml, 2.0 mmol)dropwise. The reaction mixture was stirred at rt for 2 h and quenchedwith water. The organic layer was separated, dried over Na₂SO₄, filteredand concentrated. The residue was purified by silica gel chromatography(DCM/MeOH=10/1) to give 107d (284 mg, 70%) as brown solid. MS-ESI:[M+H]⁺ 409.0

Step 5:4-(4,4-Dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-10-yl)[1,2]oxaborolo[4,3-c]pyridin-1(3H)-ol107g

4-Chloro-2-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-10-yl}pyridine-3-carbaldehyde107e was prepared following the procedures for intermediate 108a in U.S.Pat. No. 8,716,274, Example 108, Figure 8, and from4,4-dimethyl-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-9-onefrom U.S. Pat. No. 8,729,072, Example 103e. To a solution of 107e (12.0g, 35.0 mmol) in MeOH (40 mL) and DCM (40 mL) was added NaBH₄ (1.83 g,38.4 mmol) in batches at 0° C. The reaction mixture was stirred at 0° C.for 1 h, quenched with water and concentrated. The residue waspartitioned between EA and brine. The organic layer was separated, driedover Na₂SO₄, filtered and concentrated. The crude product4-Chloro-2-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.02,6]dodeca-2(6),7-dien-10-yl}pyridine-3-carbinol107f was used in next step without further purification (9.6 g, 80%). Amixture of compound 107f (9.6 g, 27.8 mmol), tetrahydroxydiborane,hypodiboric acid (7.43 g, 83.5 mmol), Xphos-Pd-G2 (218 mg, 0.278 mmol),Xphos (321 mg, 0.556 mmol) and KOAc (6.85 g, 83.5 mmol) in EtOH (200 mL)was heated to 80° C. for 1 h and concentrated. The residue was dissolvedin sat. K₂CO₃ (100 mL) and extracted with DCM 4 times. The organic phasewas discarded and the aqueous layer was neutralized with conc. HCl.White precipitate evolved and the suspension was extracted with DCM. Theorganic layer was combined, dried over Na₂SO₄, filtered and concentratedto give 107g as a gray solid (6.21 g, 53%).

Step 6: A mixture of 107d (150 mg, 0.37 mmol), 107g (125 mg, 0.37 mmol),Pd(dppf)Cl₂ (27 mg, 10 mmol %) and K₃PO₄ (235 mg, 1.11 mmol) inacetonitrile (20 mL) and water (5 ml) was stirred at 85° C. for 4 hunder nitrogen. The reaction mixture was cooled to rt and concentrated.The residue was purified by prep-TLC (DCM/MeOH=30/1) to give 107 (80 mg,34%) as a yellow solid. ¹H NMR (400 MHz, DMSO-d₆): δ 8.61-8.58 (m, 2H),8.10 (bs, 1H), 7.52-7.45 (m, 3H), 7.38-7.34 (m, 2H), 6.86 (d, J=7.6 Hz,1H), 6.51 (s, 1H), 6.15 (bs, 2H), 6.15-6.01 (m, 1H), 5.56 (bs, 1H), 4.97(bs, 1H), 4.39-4.33 (m, 2H), 4.19-4.03 (m, 6H), 3.87 (bs, 1H), 3.61 (s,3H), 2.56-2.42 (m, 4H), 1.21 (s, 6H), 0.97-0.91 (m, 3H). ESI-LCMS:m/z=638 [C35H39N7O5+H]+

Example 108N-[(1S)-2-[[6-[[5-[5-fluoro-2-(hydroxymethyl)-3-(4-oxo-6,7,8,9-tetrahydrobenzothiopheno[2,3-d]pyridazin-3-yl)phenyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]oxy]-1-methyl-ethyl]prop-2-enamide108

Step 1:(4-fluoro-2-{6-oxo-8-thia-4,5-diazatricyclo[7.4.0.02,7]trideca-1(9),2(7),3-triene-5-yl}-6-(bromo)phenyl)methylacetate 108b

8-thia-4,5-diazatricyclo[7.4.0.0^(2,7)]trideca-1(9),2(7),3-trien-6-onewas prepared following the procedures of U.S. Pat. No. 8,716,274,Example 191d and converted to(4-fluoro-2-{6-oxo-8-thia-4,5-diazatricyclo[7.4.0.0^(2,7)]trideca-1(9),2(7),3-triene-5-yl}-6-(bromo)phenyl)carbinol108a. To a mixture of 108a (4.1 g, 10 mmol) and Et₃N (1.95 mL, 14 mmol)in THF (50 mL) was added AcCl (0.85 mL, 12 mmol). The mixture wasstirred at rt for 1 h, diluted with water and extracted with EA. Thecombined organic layers were dried over Na₂SO₄, filtered andconcentrated to give compound 108b (4.0 g) as yellow solid. MS-ESI:[M+H]⁺ 451.0

Step 2:(4-fluoro-2-{6-oxo-8-thia-4,5-diazatricyclo[7.4.0.02,7]trideca-1(9),2(7),3-triene-5-yl}-6-(tetra-methyl-1,3,2-dioxaborolan-2-yl)phenyl)methylacetate 108c

A mixture of compound 108b (4.5 g, 10 mmol),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane), Pin₂B₂ (7.6g, 30 mmol), Pd(dppf)Cl₂ (378 mg, 0.5 mmol) and KOAc (2.9 g, 30 mmol) indioxane (80 mL) was stirred at 90° C. for 5 h under N₂. The reactionmixture was concentrated. The residue was slurried with PE/EA (40 mL/2mL) and filtered to give compound 108c (3.5 g, 70%) as yellow solid.MS-ESI: [M+H]⁺ 498.9

Step 3:N-[(1S)-2-[[6-[[5-[5-fluoro-2-(acetoxymethyl)-3-(4-oxo-6,7,8,9-tetrahydrobenzothiopheno[2,3-d]pyridazin-3-yl)phenyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]oxy]-1-methyl-ethyl]prop-2-enamide108d

A mixture of(S)—N-(1-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-2-yloxy)propan-2-yl)acrylamide107d (150 mg, 0.37 mmol), 108c (200 mg, 0.40 mmol), Pd(dppf)Cl₂(27 mg,10 mol %) and K₃PO₄(235 mg, 1.11 mmol) in acetonitrile (20 mL) and water(5 ml) was stirred at 85° C. for 4 h under nitrogen. The reactionmixture was cooled to rt and concentrated. The residue was purified byprep-TLC (DCM/MeOH=30/1) to give 108d (70 mg crude) as yellow solid

Step 4: To a mixture of 108d (70 mg, 0.1 mmol) in THF (6 mL), i-PrOH (2mL) and H₂O (2 mL) was added LiOH.H₂O (42 mg, 1 mmol). The reactionmixture was stirred at rt for 16 h and concentrated. The residue waspurified by prep-HPLC to give 108 (20 mg, 8% for two steps) as yellowsolid. ¹H NMR (500 MHz, DMSO-d₆): δ 8.61 (d, J=10 Hz, 2H), 8.49 (s, 1H),8.04-8.03 (m, 1H), 7.52-7.50 (m, 2H), 7.40-7.35 (m, 2H), 6.87 (d, J=8Hz, 1H), 6.21-6.15 (m, 2H), 6.05-6.02 (m, 1H), 5.54 (d, J=10 Hz, 1H),4.73 (bs, 1H), 4.30 (bs, 2H), 4.15 (bs, 2H), 4.02 (bs, 1H), 3.61 (s,3H), 2.93 (bs, 2H), 2.84 (bs, 2H), 1.86 (bs, 4H), 0.97 (bs, 3H).ESI-LCMS: m/z=657.3 [C34H33FN6O5S+H]+

Example 109N-[2-[[6-[[5-[2-(7,7-dimethyl-4-oxo-1,2,6,8-tetrahydrocyclopenta[3,4]pyrrolo[3,5-b]pyrazin-3-yl)-3-(hydroxymethyl)-4-pyridyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]amino]ethyl]prop-2-enamide109

A mixture ofN-(2-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-2-ylamino)ethyl)acrylamide105e (100 mg, 0.255 mmol),4-(4,4-Dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.02,6]dodeca-2(6),7-dien-10-yl)[1,2]oxaborolo[4,3-c]pyridin-1(3H)-ol107g (86 mg, 0.255 mmol), Pd(dppf)Cl₂(20 mg, 10 mol %), Xant-phos (30mg, 20 mol %) and K₃PO₄(160 mg, 0.765 mmol) in acetonitrile (10 mL) andH₂O (2 mL) was stirred at 85° C. for 4 h under nitrogen. The reactionmixture was concentrated and the residue was purified by prep-HPLC togive 109 (10 mg, 6%) as yellow solid. ¹H NMR (500 MHz, DMSO-d₆): δ 8.69(d, J=2.0 Hz, 1H), 8.46 (d, J=5.0 Hz, 1H), 8.22 (t, J=5.0 Hz, 1H), 8.18(s, 1H), 7.55 (s, 1H), 7.35 (d, J=5.0 Hz, 1H), 7.24 (t, J=8.0 Hz, 1H),6.57 (s, 1H), 6.44 (bs, 1H), 6.38 (d, J=8.0 Hz, 1H), 6.19-6.14 (m, 1H),6.06-6.02 (m, 1H), 5.96 (d, J=8.0 Hz, 1H), 5.55 (d, J=12.5 Hz, 1H), 5.09(bs, 1H), 4.52-4.43 (m, 2H), 4.25-4.19 (m, 3H), 3.86-3.84 (m, 1H), 3.61(s, 3H), 3.29-3.27 (m, 2H), 3.24-3.22 (m, 2H), 2.59-2.51 (m, 2H), 2.42(s, 2H), 1.22 (s, 6H). ESI-LCMS: m/z=623.0 [C34H38N8O4+H]+

Example 110N-[2-[[6-[[5-[2-(7,7-dimethyl-4-oxo-1,2,6,8-tetrahydrocyclopenta[3,4]pyrrolo[3,5-b]pyrazin-3-yl)-3-(hydroxymethyl)-4-pyridyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]-methyl-amino]ethyl]prop-2-enamide110

Step 1: A mixture of N,N-bis-Boc 6-bromopyridin-2-amine 105a (5.0 g,13.4 mmol), tert-butyl 2-(methylamino)ethylcarbamate (3.5 g, 20.0 mmol),Pd₂(dba)₃ (610 mg, 0.67 mmol), Xantphos (774 mg, 1.34 mmol) and Cs₂CO₃(10.9 g, 31.5 mmol) in DMF (50 mL) was stirred at 95° C. for 7 h underN₂. The mixture was filtered and the filtrate was diluted with EA (50mL), washed with water. The organic layer was separated, dried overNa₂SO₄, filtered and concentrated. The residue was purified by silicagel chromatography (PE/EA=9/1) to give a mixture of N,N-bis-Boc,6-(2-N-Boc-1-methylaminoethyl)pyridin-2-amine 110a (ESI-LCMS:m/z=467.3), and N-Boc, 6-(2-N-Boc-1-methylaminoethyl)pyridin-2-amine110b (ESI-LCMS: m/z=367.3), 2.0 g, 40%, as a yellow solid.

Step 2: A mixture of 110a and 110b (2.0 g, 4.3 mmol) in HCl (10 mL, 4 Min 1,4-dioxane, 40 mmol) was stirred at rt for 7 h. The reaction mixturewas concentrated to give tert-butyl6-((2-aminoethyl)(methyl)amino)pyridin-2-ylcarbamate hydrochloride 110c(1.2 g, 93%) as yellow solid which was used for the next step directly.ESI-LCMS: m/z=267.2

Step 3: A mixture of 110c (1.2 g, 4.5 mmol), acrylic acid (0.37 mL, 5.4mmol), HATU (2.05 g, 5.4 mmol) and DIPEA (4.6 mL, 10.8 mmol) in DCM (30mL) was stirred at rt overnight. The mixture was washed with water. Theorganic layer was separated, dried over Na₂SO₄, filtered andconcentrated to give tert-butyl6-((2-acrylamidoethyl)(methyl)amino)pyridin-2-ylcarbamate 110d (1.2 g,75%) as a brown oil. The material was used for the next step directly.ESI-LCMS: m/z=321.3

Step 4: A mixture of 110d (2.0 g, 6.25 mmol) and TFA (2.0 mL) in DCM(10.0 mL) was stirred at rt overnight. The mixture was concentrated anddiluted with EA (30 mL), neutralized with 1M NaOH. The organic layer wasseparated, dried over Na₂SO₄, filtered and concentrated. The residue waspurified by prep-TLC (PE/EA=1/1) to giveN-(2-((6-aminopyridin-2-yl)(methyl)amino)ethyl)acrylamide 110e (550 mg,40%) as yellow oil. ESI-LCMS: m/z=221.2

Step 5: A mixture of 110e (400 mg, 1.82 mmol),3,5-dibromo-1-methylpyridin-2(1H)-one (534 mg, 2.0 mmol), Pd₂(dba)₃ (83mg, 0.091 mmol), Xantphos (105 mg, 0.182 mmol) and Cs₂CO₃ (1.48 g, 4.55mmol) in DMF (10 mL) was stirred at 95° C. under N₂ for 7 h. The mixturewas diluted with EA (50 mL) and filtered. The filtrate was washed withwater, dried over Na₂SO₄, filtered and concentrated. The residue waspurified by silica gel chromatography (PE/EA=1/2) to giveN-(2-((6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-2-yl)(methyl)amino)ethyl)acrylamide110f (160 mg, 22%) as a yellow solid. ESI-LCMS: m/z=408.0

Step 6: A mixture of 110f (80 mg, 0.20 mmol),4-(4,4-Dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.02,6]dodeca-2(6),7-dien-10-yl)[1,2]oxaborolo[4,3-c]pyridin-1(3H)-ol107g (83 mg, 0.22 mmol), Pd(dppf)Cl₂ (15 mg, 0.02 mmol) and K₃PO₄ (106mg, 0.5 mmol) in acetonitrile (5 mL) and water (1 mL) was stirred at 90°C. under N₂ for 7 h. The mixture was filtered and the filtrate wasconcentrated. The residue was purified by prep-TLC (EA/MeOH=20/1) togive 110 (60 mg, 48%) as light yellow solid. ¹H NMR (400 MHz, CDCl₃): δ8.63 (s, 1H), 8.46-8.45 (d, J=4.8 Hz, 1H), 7.81 (s, 1H), 7.75 (s, 1H),7.41-7.34 (m, 1H), 6.83 (s, 1H), 6.65 (s, 1H), 6.19-6.18 (d, J=7.2 Hz,1H), 6.03-5.95 (m, 2H), 5.72-5.65 (m, 1H), 5.42-5.39 (d, J=10.4 Hz, 1H),4.96-3.98 (m, 7H), 3.71 (s, 3H), 3.53-3.22 (m, 3H), 2.99 (s, 3H), 2.57(s, 2H), 2.51 (s, 2H), 1.27 (s, 6H). ESI-LCMS: m/z=637 [C35H40N8O4+H]+

Example 111N-[2-[[6-[[5-[5-fluoro-2-(hydroxymethyl)-3-(4-oxo-6,7,8,9-tetrahydrobenzothiopheno[2,3-d]pyridazin-3-yl)phenyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]-methyl-amino]ethyl]prop-2-enamide111

Step 1: A mixture ofN-(2-((6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-2-yl)(methyl)amino)ethyl)acrylamide110f (120 mg, 0.3 mmol),(4-fluoro-2-{6-oxo-8-thia-4,5-diazatricyclo[7.4.0.02,7]trideca-1(9),2(7),3-triene-5-yl}-6-(tetra-methyl-1,3,2-dioxaborolan-2-yl)phenyl)methylacetate 108c (179 mg, 0.36 mmol), Pd(dppf)Cl₂ (23 mg, 0.03 mmol) andK₃PO₄ (159 mg, 0.75 mmol) in acetonitrile (5 mL) and water (1.0 mL) wasstirred at 90° C. under N₂ for 7 h. The mixture was filtered and thefiltrate was concentrated. The residue was purified by prep-TLC(EA/MeOH=25/1) to giveN-[2-[[6-[[5-[5-fluoro-2-(acetoxymethyl)-3-(4-oxo-6,7,8,9-tetrahydrobenzothiopheno[2,3-d]pyridazin-3-yl)phenyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]-methyl-amino]ethyl]prop-2-enamide111a (150 mg, 60%) as a light yellow solid. ESI-LCMS: m/z=698.2

Step 2: A mixture of 111a (130 mg, 0.186 mmol) and LiOH.H₂O (40 mg, 0.93mmol) in THF/i-PrOH/water (2.0 mL/1.0 mL/1.0 mL) was stirred at rt for 2h. The mixture was extracted with EA. The combined organic layers weredried over Na₂SO₄, filtered and concentrated. The residue was purifiedby prep-TLC (DCM/MeOH=20/1) to give 111 (40 mg, 33%) as white solid. ¹HNMR (400 MHz, CDCl₃): δ 8.53 (s, 1H), 8.25 (s, 1H), 7.77 (s, 1H), 7.47(s, 1H), 7.39-7.35 (t, J=8.4 Hz, 1H), 7.26-7.23 (m, 1H), 7.10-7.08 (m,1H), 6.64 (s, 1H), 6.18-6.16 (d, J=8.4 Hz, 1H), 6.08-6.00 (m, 2H),5.85-5.79 (m, 1H), 5.47-5.44 (d, J=10.4 Hz, 1H), 4.30-4.28 (d, J=6.4 Hz,2H), 4.02-3.99 (t, J=6.4 Hz, 1H), 3.75-3.69 (m, 5H), 3.47-3.45 (m, 2H),3.02-2.98 (m, 5H), 2.86 (m, 2H), 1.98 (m, 4H). ESI-LCMS: m/z=656[C34H34FN7O4S+H]+

Example 112N-[2-[[6-[[5-[2-(7,7-dimethyl-4-oxo-1,2,6,8-tetrahydrocyclopenta[3,4]pyrrolo[3,5-b]pyrazin-3-yl)-3-(hydroxymethyl)-4-pyridyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]oxy]ethyl]-N-methyl-prop-2-enamide112

Step 1: To a mixture of 2-(methylamino)ethanol (5.0 g, 66.6 mmol) in DCM(100 mL) was added a solution of (Boc)₂O (15.0 g, 67.9 mmol) in DCM (20mL). The mixture was stirred at rt for 2 h, diluted with EA (100 mL) andwashed with water. The organic layer was separated, dried over Na₂SO₄,filtered and concentrated to give tert-butyl2-hydroxyethyl(methyl)carbamate 112a (10.0 g, 86%) as colorless oil.

Step 2: To a mixture of 112a (9.0 g, 51 mmol), and Et₃N (18.6 mL, 51mmol) in DCM (100 mL) was added TsCl (9.77 g, 51 mmol). The mixture wasstirred at rt for 4 h and concentrated. The residue was diluted with EA,washed with water. The organic layer was dried and concentrated to give2-(tert-butoxycarbonyl(methyl)amino)ethyl 4-methylbenzenesulfonate 112b(10.0 g, 61%) as light yellow oil. ESI-LCMS: m/z=230.0

Step 3: A mixture of 112b (3.3 g, 10 mmol),5-Bromo-3-(6-hydroxypyridin-2-ylamino)-1-methylpyridin-2(1H)-one 101a(7.4 g, 25 mmol) and Cs₂CO₃ (4.1 g, 12.5 mmol) in DMF (40 mL) wasstirred at 100° C. for 4 h. The mixture partitioned between EA andwater. The organic layer was separated, dried over Na₂SO₄, filtered andconcentrated to give tert-butyl2-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-2-yloxy)ethyl(methyl)carbamate112c (1.0 g, 22%) as a yellow solid. ESI-LCMS: m/z=454.9

Step 4: A mixture of 112c (900 mg, 1.99 mmol) in HCl (5 mL, 4 M in1,4-dioxane, 20 mmol) was stirred at rt for 2 h. The reaction mixturewas concentrated to give5-bromo-1-methyl-3-(6-(2-(methylamino)ethoxy)pyridin-2-ylamino)pyridin-2(1H)-onehydrochloride 112d (660 mg, 85%) as yellow solid. This material was usedfor next step directly. ESI-LCMS: m/z=354.9 (free base)

Step 5: A mixture of 112d (800 mg, 2.27 mmol) and Et₃N (0.79 mL, 5.67mmol) in DCM (20 mL) was added acryloyl chloride (0.23 mL, 2.84 mmol).The mixture was stirred at rt or 2 h and quenched with water. Theorganic layer was separated, dried over Na₂SO₄, filtered andconcentrated to giveN-(2-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-2-yloxy)ethyl)-N-methylacrylamide112e (250 mg, 27%) as light yellow solid. ESI-LCMS: m/z=407.0

Step 6: A mixture of 112e (200 mg, 0.5 mmol),4-(4,4-Dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.02,6]dodeca-2(6),7-dien-10-yl)[1,2]oxaborolo[4,3-c]pyridin-1(3H)-ol107g (185 mg, 0.55 mmol), Pd(dppf)Cl₂ (37 mg, 0.05 mmol) and K₃PO₄ (265mg, 1.25 mmol) in acetonitrile (5 mL) and water (1 mL) was stirred at90° C. for 16 h under N₂. The mixture was filtered and the filtrate wasconcentrated. The residue was purified by prep-TLC (EA/MeOH=25/1) togive 112 (70 mg, 22%) as light yellow solid. ¹H NMR (300 MHz, DMSO-d₆,80° C.): δ 8.47-8.45 (m, 2H), 8.34 (s, 1H), 7.51 (t, J=7.5 Hz, 2H), 7.32(d, J=5.1 Hz, 1H), 6.80 (d, J=5.1 Hz, 1H), 6.69-6.58 (m, 1H), 6.55 (s,1H), 6.19 (d, J=7.8 Hz, 1H), 6.03-5.97 (m, 1H), 5.53 (bs, 1H), 4.76 (t,J=5.1 Hz, 1H), 4.48-4.46 (m, 2H), 4.32 (t, J=5.4 Hz, 2H), 4.21-4.17 (m,2H), 3.67 (t, J=5.1 Hz, 2H), 3.61 (s, 3H), 3.04 (s, 3H), 2.88 (bs, 2H),2.57 (s, 2H), 2.44 (s, 2H), 1.23 (s, 6H). ESI-LCMS: m/z=638.0[C35H39N7O5+H]+

Example 113N-[2-[[6-[[5-[5-fluoro-2-(hydroxymethyl)-3-(4-oxo-6,7,8,9-tetrahydrobenzothiopheno[2,3-d]pyridazin-3-yl)phenyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]oxy]ethyl]-N-methyl-prop-2-enamide113

Step 1: A mixture ofN-(2-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-2-yloxy)ethyl)-N-methylacrylamide112e (100 mg, 0.25 mmol),(4-fluoro-2-{6-oxo-8-thia-4,5-diazatricyclo[7.4.0.02,7]trideca-1(9),2(7),3-triene-5-yl}-6-(tetra-methyl-1,3,2-dioxaborolan-2-yl)phenyl)methylacetate 108c (187 mg, 0.375 mmol), Pd(dppf)Cl₂ (19 mg, 0.025 mmol) andK₃PO₄ (133 mg, 0.625 mmol) in acetonitrile (5 mL) and water (0.5 mL) wasstirred at 90° C. for 6 h under N₂. The mixture was filtered and thefiltrate was concentrated. The residue was purified by prep-TLC(PE/EA=1/4) to giveN-[2-[[6-[[5-[5-fluoro-2-(acetoxymethyl)-3-(4-oxo-6,7,8,9-tetrahydrobenzothiopheno[2,3-d]pyridazin-3-yl)phenyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]oxy]ethyl]-N-methyl-prop-2-enamide113a (100 mg, 57%) as light yellow solid. MS-ESI: [M+H]⁺ 699.2

Step 2: A mixture of 113a (80 mg, 0.115 mmol) and LiOH.H₂O (24 mg, 0.573mmol) in THF/i-PrOH/water (3.0 mL/1.0 mL/1.0 mL) was stirred at rt for 6h. The mixture was extracted with EA. The combined organic layers weredried over Na₂SO₄, filtered and concentrated. The residue was purifiedby prep-TLC (EA/MeOH=25/1) to give 113 (50 mg, 67%) as white solid. ¹HNMR (300 MHz, DMSO-d₆, 80° C.): δ 8.42 (s, 2H), 8.35 (s, 1H), 7.52 (t,J=8.1 Hz, 1H), 7.43 (d, J=2.4 Hz, 1H), 7.33-7.30 (m, 2H), 6.81 (d, J=8.1Hz, 1H), 6.68-6.59 (m, 1H), 6.19 (d, J=8.1 Hz, 1H), 6.05-5.99 (m, 1H),5.56 (m, 1H), 4.37-4.32 (m, 2H), 3.68 (t, J=5.4 Hz, 2H), 3.62 (s, 3H),3.07 (s, 3H), 2.95-2.86 (m, 6H), 1.91-1.89 (m, 4H). ESI-LCMS: m/z=657.3[C34H33FN6O5S+H]+

Example 114N-[(3S)-1-[6-[[5-[2-(7,7-dimethyl-4-oxo-1,2,6,8-tetrahydrocyclopenta[3,4]pyrrolo[3,5-b]pyrazin-3-yl)-3-(hydroxymethyl)-4-pyridyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]-3-piperidyl]prop-2-enamide114

Step 1: A mixture of N,N-bis-Boc 6-bromopyridin-2-amine 105a (5.0 g,13.4 mmol), (5)-tert-butyl piperidin-3-ylcarbamate (4 g, 20.0 mmol),Pd₂(dba)₃ (610 mg, 0.67 mmol), Xantphos (774 mg, 1.34 mmol) and Cs₂CO₃(10.9 g, 31.5 mmol) in DMF (50 mL) was stirred at 95° C. for 7 h underN₂. The mixture was filtered and the filtrate was diluted with EA (50mL), washed with water. The organic layer was separated, dried overNa₂SO₄, filtered and concentrated. The residue was purified by silicagel chromatography (PE/EA=9/1) to give a mixture of(S)—N—Boc-6-(3-(bis-Boc-amino)piperidin-1-yl)pyridin-2-amine 114a and(S)—N—Boc-6-(3-(Boc-amino)piperidin-1-yl)pyridin-2-amine 114b (2.3 g,35%) as yellow solid.

Step 2: A mixture of 114a and 114b (2.3 g, 4.69 mmol) in HCl (10 mL, 4 Min 1,4-dioxane, 40 mmol) was stirred at rt for 7 h. The reaction mixturewas concentrated to give (S)-tert-butyl6-(3-aminopiperidin-1-yl)pyridin-2-ylcarbamate hydrochloride 114c (1.39g, 90%) as yellow solid which was used for the next step directly.

Step 3: A mixture of 114c (1.39 g, 4.22 mmol), acrylic acid (0.3 mL, 4.5mmol), HATU (1.7 g, 4.5 mmol) and DIPEA (3.8 mL, 9 mmol) in DCM (30 mL)was stirred at rt overnight. The mixture was washed with water. Theorganic layer was separated, dried over Na₂SO₄, filtered andconcentrated to give (S)-tert-butyl6-(3-acrylamidopiperidin-1-yl)pyridin-2-ylcarbamate 114d (880 mg, 60%)as brown oil. The material was used for the next step directly.

Step 4: A mixture of 114d (880 mg, 2.53 mmol) and TFA (2.0 mL) in DCM(10.0 mL) was stirred at rt overnight. The mixture was concentrated anddiluted with EA (30 mL), neutralized with 1M NaOH. The organic layer wasseparated, dried over Na₂SO₄, filtered and concentrated. The residue waspurified by prep-TLC (PE/EA=1/1) to give(S)—N-(1-(6-aminopyridin-2-yl)piperidin-3-yl)acrylamide 114e (440 mg,70%) as yellow oil. MS-ESI: [M+H]⁺ 247.2

Step 5: A mixture of 114e (440 mg, 1.79 mmol),3,5-dibromo-1-methylpyridin-2(1H)-one (534 mg, 2.0 mmol), Pd₂(dba)₃ (83mg, 0.091 mmol), Xantphos (105 mg, 0.182 mmol) and Cs₂CO₃ (1.48 g, 4.55mmol) in DMF (10 mL) was stirred at 95° C. under N₂ for 7 h. The mixturewas diluted with EA and filtered. The filtrate was washed with water,dried over Na₂SO₄, filtered and concentrated. The residue was purifiedby silica gel chromatography (PE/EA=1/2) to give(S)—N-(1-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-2-yl)piperidin-3-yl)acrylamide114f (220 mg, 28%) as yellow solid. MS-ESI: [M+H]⁺ 433.3

Step 6: A mixture of 114f (86 mg, 0.20 mmol),4-(4,4-Dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.02,6]dodeca-2(6),7-dien-10-yl)[1,2]oxaborolo[4,3-c]pyridin-1(3H)-ol107g (83 mg, 0.22 mmol), Pd(dppf)Cl₂ (15 mg, 0.02 mmol) and K₃PO₄ (106mg, 0.5 mmol) in acetonitrile (5 mL) and water (1 mL) was stirred at 90°C. under N₂ for 7 h. The mixture was filtered and the filtrate wasconcentrated. The residue was purified by prep-HPLC to give 114 (25 mg,19%) as gray solid. ¹H NMR (300 MHz, DMSO-d₆, 80° C.): δ 8.39 (s, 1H),8.00 (s, 1H), 7.82 (d, J=6.6 Hz, 1H), 7.32-7.46 (m, 6H), 6.51 (s, 1H),6.42 (d, J=8.1 Hz, 1H), 6.03-6.24 (m, 3H), 5.54 (d, J=13.5 Hz, 1H), 4.65(bs, 1H), 4.41 (bs, 2H), 4.21-4.07 (m, 3H), 3.94-3.77 (m, 4H), 3.60 (s,3H), 2.95 (bs, 2H), 2.58 (s, 2H), 2.44 (s, 2H), 1.85 (bs, 1H), 1.63-1.48(m, 3H), 1.24 (s, 6H). ESI-LCMS: m/z=663 [C37H42N8O4+H]+

Example 115N-[(3S)-1-[6-[[5-[5-fluoro-2-(hydroxymethyl)-3-(4-oxo-6,7,8,9-tetrahydrobenzothiopheno[2,3-d]pyridazin-3-yl)phenyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]-3-piperidyl]prop-2-enamide115

Step 1: A mixture of(S)—N-(1-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-2-yl)piperidin-3-yl)acrylamide114f (127 mg, 0.3 mmol),(4-fluoro-2-{6-oxo-8-thia-4,5-diazatricyclo[7.4.0.02,7]trideca-1(9),2(7),3-triene-5-yl}-6-(tetra-methyl-1,3,2-dioxaborolan-2-yl)phenyl)methylacetate 108c (179 mg, 0.36 mmol), Pd(dppf)Cl₂ (23 mg, 0.03 mmol) andK₃PO₄ (159 mg, 0.75 mmol) in acetonitrile (5 mL) and water (1.0 mL) wasstirred at 90° C. under N₂ for 7 h. The mixture was filtered and thefiltrate was concentrated. The residue was purified by prep-TLC(EA/MeOH=25/1) to giveN-[(3S)-1-[6-[[5-[5-fluoro-2-(acetoxymethyl)-3-(4-oxo-6,7,8,9-tetrahydrobenzothiopheno[2,3-d]pyridazin-3-yl)phenyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]-3-piperidyl]prop-2-enamide115a (73 mg, 33%) as yellow solid. MS-ESI: [M+H]⁺ 724.3

Step 2: A mixture of 115a (73 mg, 0.1 mmol) and LiOH.H₂O (40 mg, 1 mmol)in THF/i-PrOH/water (2.0 mL/1.0 mL/1.0 mL) was stirred at rt for 2 h.The mixture was extracted with EA. The combined organic layers weredried over Na₂SO₄, filtered and concentrated. The residue was purifiedby prep-HPLC to give 115 (20 mg, 29%) as gray solid. ¹H NMR (300 MHz,DMSO-d₆, 80° C.): δ 8.42 (s, 1H), 8.40 (s, 1H), 8.00 (s, 1H), 7.77 (d,J=4.5 Hz, 1H), 7.41-7.29 (m, 5H), 6.44 (d, J=3 Hz, 1H), 6.25-6.17 (m,2H), 5.55-5.50 (m, 1H), 4.35 (s, 2H), 3.98-3.76 (m, 3H), 3.61 (s, 3H),3.09-2.87 (m, 7H), 1.93-1.87 (m, 6H), 1.71 (bs, 1H), 1.49-1.45 (m, 2H).ESI-LCMS: m/z=682 [C36H36FN7O4S+H]+

Example 116N-[2-[[6-[[5-[2-(7,7-dimethyl-4-oxo-1,2,6,8-tetrahydrocyclopenta[3,4]pyrrolo[3,5-b]pyrazin-3-yl)-3-(hydroxymethyl)-4-pyridyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]oxy]ethyl]but-2-ynamide116

Step 1: A mixture of3-(6-(2-aminoethoxyl)pyridin-2-ylamino)-5-bromo-1-methylpyridin-2(1H)-onehydrochloride 103a (1.0 g, 2.66 mmol), but-2-ynoic acid (268 mg, 3.19mmol), HATU (1.21 g, 3.19 mmol) and DIPEA (1.14 mL, 6.65 mmol) in DMF(10 mL) was stirred at rt overnight. The reaction mixture was dilutedwith EA and washed with water. The organic layer was separated, driedover Na₂SO₄, filtered and concentrated. The residue was washed withPE/EA (2 mL/20 mL) and dried to giveN-(2-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-2-yloxy)ethyl)but-2-ynamide116a (800 mg, 74%) as light yellow solid. ESI-LCMS: m/z=404.9

Step 2: A mixture of 116a (150 mg, 0.37 mmol),4-(4,4-Dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.02,6]dodeca-2(6),7-dien-10-yl)[1,2]oxaborolo[4,3-c]pyridin-1(3H)-ol107g (150 mg, 0.445 mmol), Pd(dppf)Cl₂ (28 mg, 0.037 mmol) and K₃PO₄(196 mg, 0.925 mmol) in acetonitrile (5 mL) and water (1 mL) was stirredat 90° C. for 16 h under N₂. The mixture was filtered and the filtratewas concentrated. The residue was purified by prep-HPLC to give 116 (20mg, 10%) as light yellow solid. ¹H NMR (500 MHz, CDCl₃): δ 8.66 (s, 1H),8.56-8.55 (m, 1H), 7.94 (s, 1H), 7.85 (s, 1H), 7.48 (t, J=8.0 Hz, 1H),7.38 (s, 1H), 6.87 (s, 1H), 6.49 (s, 1H), 6.44 (d, J=8.0 Hz, 1H), 6.26(d, J=8.0 Hz, 1H), 5.05-3.97 (m, 8H), 3.72 (s, 3H), 3.59-3.57 (m, 2H),2.57 (s, 2H), 2.51 (s, 2H), 1.91 (s, 3H), 1.27 (s, 6H). ESI-LCMS:m/z=636.9 [C35H37N7O5+H]+

Example 1172-cyano-N-[2-[[6-[[5-[5-fluoro-2-(hydroxymethyl)-3-(4-oxo-6,7,8,9-tetrahydrobenzothiopheno[2,3-d]pyridazin-3-yl)phenyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]oxy]ethyl]prop-2-enamide117

Step 1: A mixture of3-(6-(2-aminoethoxyl)pyridin-2-ylamino)-5-bromo-1-methylpyridin-2(1H)-one103a (480 mg, 1.42 mmol), 2-cyanoacrylic acid (790 mg, 2.84 mmol), DIPEA(1.2 mL, 7.12 mmol) and HATU (810 mg, 2.13 mmol) in DCM (10 mL) wasstirred at rt for 16 h. The reaction mixture was concentrated andpurified by silica gel chromatography (PE/EA=1/2) to giveN-(2-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-2-yloxy)ethyl)-2-cyanoacrylamide117a (400 mg, 67%) as green solid. ESI-LCMS: m/z=420.0

Step 2: A mixture of 117a (300 mg, 0.72 mmol),(4-fluoro-2-{6-oxo-8-thia-4,5-diazatricyclo[7.4.0.02,7]trideca-1(9),2(7),3-triene-5-yl}-6-(tetra-methyl-1,3,2-dioxaborolan-2-yl)phenyl)methylacetate 108c (430 mg, 0.86 mmol), Pd(dppf)Cl₂ (52 mg, 0.072 mmol), KF(125 mg, 2.16 mmol) in acetonitrile (10 mL) and H₂O (2 mL) was stirredat 75° C. for 3 h under nitrogen. The reaction mixture was concentratedand the residue was purified by silica gel chromatography (PE/EA=1/3) togive2-cyano-N-[2-[[6-[[5-[5-fluoro-2-(acetoxymethyl)-3-(4-oxo-6,7,8,9-tetrahydrobenzothiopheno[2,3-d]pyridazin-3-yl)phenyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]oxy]ethyl]prop-2-enamide117b (80 mg, 15%) as white solid. ESI-LCMS: m/z=709.8

Step 3: To a mixture of 117b (80 mg, 0.112 mmol) in THF (5 mL) was addedLiOH.H₂O (10 mg, 0.224 mmol) in H₂O (5 mL) to pH 10-11. The reactionmixture was stirred at rt for 4 h and concentrated. The residue waspurified by prep-HPLC to give 117 (12 mg, 16%) as white solid. The amidebond was partially cleaved under basic conditions. ¹H NMR (500 MHz,CDCl₃): δ8.57 (s, 1H), 8.29 (s, 1H), 7.99 (bs, 1H), 7.49 (t, J=8 Hz,2H), 7.26-7.24 (m, 1H), 7.12 (d, J=10 Hz, 1H), 6.48 (d, J=8 Hz, 1H),6.26 (d, J=8 Hz, 1H), 4.41 (t, J=5 Hz, 2H), 4.31 (bs, 2H), 4.00-3.98 (m,1H), 3.72 (s, 3H), 3.63 (t, J=5.5 Hz, 1H), 3.58-2.56 (m, 1H), 2.99 (t,J=5 Hz, 2H), 2.87 (t, J=5 Hz, 2H), 2.00-1.96 (m, 4H). ESI-LCMS:m/z=668.0 [C34H30FN7O5S+H]+

Example 1182-cyano-N-[2-[[6-[[5-[2-(7,7-dimethyl-4-oxo-1,2,6,8-tetrahydrocyclopenta[3,4]pyrrolo[3,5-b]pyrazin-3-yl)-3-(hydroxymethyl)-4-pyridyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]oxy]ethyl]prop-2-enamide118

A mixture ofN-(2-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-2-yloxy)ethyl)-2-cyanoacrylamide117a (60 mg, 0.144 mmol),444,4-Dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.02,6]dodeca-2(6),7-dien-10-yl)[1,2]oxaborolo[4,3-c]pyridin-1(3H)-ol107g (58 mg, 0.173 mmol), Pd(dppf)Cl₂ (11 mg, 0.0144 mmol) and KF (25mg, 0.432 mmol) in acetonitrile (5 mL) and water (0.5 mL) was stirred at75° C. for 6 h under N₂. The mixture was filtered and the filtrate wasconcentrated. The residue was purified by prep-TLC with PE/EA (1/5) togive 118 (20 mg, 22%) as light yellow solid. ¹H NMR (300 MHz, DMSO-d₆,80° C.): δ 8.43 (s, 1H), 8.34 (s, 1H), 7.56-7.32 (m, 6H), 6.81 (d, J=7.5Hz, 1H), 6.53 (s, 1H), 6.23 (d, J=7.5 Hz, 1H), 4.66-3.99 (m, 8H), 3.62(s, 3H), 3.52-3.43 (m, 3H), 2.58 (s, 2H), 2.45 (s, 2H), 1.24 (s, 6H).ESI-LCMS: m/z=649 [C35H36N8O5+H]+

Example 119N-{2-[(6-{[5-(2-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-10-yl}-3-(hydroxymethyl)pyridin-4-yl)-1-methyl-2-oxo-1,2-dihydropyridin-3-yl]amino}pyridin-2-yl)oxy]ethyl}propanamide119

Step 1: A mixture of3-(6-(2-aminoethoxyl)pyridin-2-ylamino)-5-bromo-1-methylpyridin-2(1H)-onehydrochloride 103a (250 mg, 0.66 mmol) and Et₃N (0.23 mL, 6.0 mmol) inDCM (15 mL) was added propionyl chloride (70 uL, 0.79 mmol). The mixturewas stirred at RT for 2 h. The mixture was washed with water (20 mL*2).The organic layer was dried and concentrated to giveN-(2-(6-(5-bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-2-yloxy)ethyl)propionamide119a (140 mg, 54%) as a light yellow solid. ESI-LCMS: m/z=395.1.

Step 2: A mixture of 119a (120 mg, 0.30 mmol), compound 9 (111 mg, 0.33mmol), Pd(dppf)Cl₂ (23 mg, 0.03 mmol) and K₃PO₄ (160 mg, 0.75 mmol) inCH₃CN (5 mL) and water (1 mL) was stirred at 90° C. under N₂ for 7 h.The mixture was filtered and the filtrate was concentrated to get thecrude product. It was purified by prep-HPLC to give 119 (40 mg, 21%) asa white solid. ESI-LCMS: m/z=626.3

Example 901 Biochemical Btk Assays

A generalized procedure for a standard biochemical Btk, Kinase Assaythat can be used to test Formula I compounds is as follows. A master mixminus Btk enzyme is prepared containing 1× Cell Signaling kinase buffer(25 mM Tris-HCl, pH 7.5, 5 mM beta-glycerophosphate, 2 mMdithiothreitol, 0.1 mM Na₃VO₄, 10 mM MgCl₂), 0.5 μM Promega PTKBiotinylated peptide substrate 2, and 0.01% BSA. A master mix plus Btkenzyme is prepared containing 1× Cell Signaling kinase buffer, 0.5 μMPTK Biotinylated peptide substrate 2, 0.01% BSA, and 100 ng/well (0.06mU/well) Btk enzyme. Btk enzyme is prepared as follows: full lengthhuman wildtype Btk (accession number NM-000061) with a C-terminal V5 and6×His tag was subcloned into pFastBac® vector (Invitrogen/LifeTechnologies) for making baculovirus carrying this epitope-tagged Btk.Generation of baculovirus is done based on Invitrogen's instructionsdetailed in its published protocol “Bac-to-Bac Baculovirus ExpressionSystems” (Invitrogen/Life Technologies, Cat. Nos. 10359-016 and10608-016). Passage 3 virus is used to infect Sf9 cells to overexpressthe recombinant Btk protein. The Btk protein is then purified tohomogeneity using Ni-NTA column. The purity of the final proteinpreparation is greater than 95% based on the sensitive Sypro-Rubystaining. A solution of 200 μM ATP is prepared in water and adjusted topH 7.4 with 1N NaOH. A quantity of 1.25 μL, of compounds in 5% DMSO istransferred to a 96-well ½ area Costar polystyrene plate. Compounds aretested singly and with an 11-point dose-responsive curve (startingconcentration is 10 μM; 1:2 dilution). A quantity of 18.75 μL, of mastermix minus enzyme (as a negative control) and master mix plus enzyme istransferred to appropriate wells in 96-well ½ area costar polystyreneplate. 5 μL, of 200 μM ATP is added to that mixture in the 96-well ½area Costar polystyrene plate for final ATP concentration of 40 μM. Thereaction is allowed to incubate for 1 hour at room temperature. Thereaction is stopped with Perkin Elmer 1× detection buffer containing 30mM EDTA, 20 nM SA-APC, and 1 nM PT66 Ab. The plate is read usingtime-resolved fluorescence with a Perkin Elmer Envision using excitationfilter 330 nm, emission filter 665 nm, and 2^(nd) emission filter 615nm. IC₅₀ values are subsequently calculated. Alternatively, theLanthascreen assay can be used to evaluate Btk activity throughquantification of its phosphorylated peptide product. The FRET(Fluorescence Resonance Energy Transfer) that occurs between thefluorescein on the peptide product and the terbium on the detectionantibody decreases with the addition of inhibitors of Btk that reducethe phosphorylation of the peptide. In a final reaction volume of 25 μL,Btk (h) (0.1 ng/25 μl reaction) is incubated with 50 mM Hepes pH 7.5, 10mM MgCl2, 2 mM MnCl₂, 2 mM DTT, 0.2 mM NaVO4, 0.01% BSA, and 0.4 μMfluorescein poly-GAT. The reaction is initiated by the addition of ATPto 25 μM (Km of ATP). After incubation for 60 minutes at roomtemperature, the reaction is stopped by the addition of a finalconcentration of 2 nM Tb-PY20 detection antibody in 60 mM EDTA for 30minutes at room temperature. Detection is determined on a Perkin ElmerEnvision with 340 nM excitation and emission at 495 nm and 520 nm.

Alternatively, an in vitro BTK biochemical assay that quantifiesBTK-catalyzed tyrosine phosphorylation of a synthetic peptide, asdetected using a LabChip 3000® microfluidic mobility shift instrument(PerkinElmer; Waltham, Mass.) can be conducted. The substrate peptide,ProfilerPro® FL-Peptide 22 (Product No. 760366; PerkinElmer), has anamino-terminal fluorescent 5-carboxyfluorescein group (5-FAM) and atyrosine residue that can be phosphorylated by BTK:5-FAM-EEPLYWSFPAKKK-NH₂. Purified recombinant human full-lengthcatalytically active BTK protein was obtained from Carna Biosciences(Product No. 08-080; Kobe, Japan).

BTK assay mixtures contained 50 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (pH 7.5), 10mM magnesium chloride, 0.01% Triton X-100, 1 mM dithiothreitol, 1 μMFL-Peptide 22, 45 μM ATP, 1 nM BTK, and a titration of up to 10,000 nMtest article in a final concentration of 0.5% (volume to volume [v/v])DMSO. In the titration experiments, each of the test articleconcentrations (10 or 12 concentrations) was tested in duplicate. Blankreactions contained ATP, peptide, and DMSO, but no BTK or test article,whereas uninhibited control reactions contained ATP, peptide, BTK, andDMSO, but no test article.

Reactions were incubated for 30 minutes at room temperature (22° C.-23°C.) in a final volume of 20 μL per well in 384-well plates. Tenmicroliters of BTK plus peptide mixture were added to 10 μL of a mixtureof ATP and test article (or vehicle) to initiate the reactions.Reactions were stopped by adding 10 μL of 0.25 Methylenediaminetetraacetic acid (EDTA) at pH 8.0 to each well. In eachreaction, the residual FL-Peptide 22 substrate (S) and thephospho-peptide product (P) generated were separated using the LabChip3000 instrument. Electrophoretic separation of molecules of product frommolecules of substrate was achieved using downstream and upstreamvoltages of −500 and −2250 V, respectively, at an operating pressure of−1 psi. The 5-FAM group present on both the substrate and productpeptides was excited at 488 nm; the fluorescence at 530 nm was detectedand the peak heights were reported.

Data Analysis: The extent (or percent) of conversion of substrate toproduct was calculated from the corresponding peak heights in theelectropherogram using HTS Well Analyzer software, version 5.2(PerkinElmer) and the following equation:conversion=[(P)÷(S+P)]×100where S and P represent the peak heights of the substrate and product,respectively. After any baseline signal from blank wells containing noBTK was subtracted from the signal of all test wells, the % conversiondata were converted to fractional activity as shown in Equation 2, wherev_(i) and v_(o) are the % conversion in the presence and absence,respectively, of test article. The % conversion observed in theuninhibited control reaction wells containing BTK and DMSO vehicle, butno test article, was defined to have fractional activity=1 (in this casewith no test article present, v_(i)=v_(o)), whereas blank wells with noBTK were defined as having fractional activity=0. Fractional activitywas plotted against test article concentration and the data were fitusing Genedata Screener software (Genedata; Basel, Switzerland) to thetight-binding apparent inhibition constant (K_(i) ^(app)) Morrisonequation [Williams, J. W. and Morrison, J. F. (1979) The kinetics ofreversible tight-binding inhibition. Methods Enzymol 63:437-67]. Thefollowing equation was used to calculate fractional activity and K_(i)^(app):

${{Fractional}\mspace{14mu}{activity}} = {\frac{v_{i}}{v_{o}} = {1 - \frac{\begin{matrix}{\left( {\lbrack E\rbrack_{T} + \lbrack I\rbrack_{T} + K_{i}^{app}} \right) -} \\\sqrt{\left( {\lbrack E\rbrack_{T} + \lbrack I\rbrack_{T} + K_{i}^{app}} \right)^{2} - {{4\lbrack E\rbrack}_{T}\lbrack I\rbrack}_{T}}\end{matrix}}{{2\lbrack E\rbrack}_{T}}}}$where [E]_(T) and [I]_(T) are the total concentrations of active enzyme(fixed value of 0.001 μM=1 nM) and test article (e.g., inhibitor; thevaried parameter), respectively.

Exemplary Btk inhibition IC50 values are in Table 1.

Example 902 Ramos Cell Btk Assay

Another generalized procedure for a standard cellular Btk, Kinase Assaythat can be used to test Formula I compounds is as follows. Ramos cellsare incubated at a density of 0.5×10⁷ cells/ml in the presence of testcompound for 1 hr at 37° C. Cells are then stimulated by incubating with10 μg/ml anti-human IgM F(ab)₂ for 5 minutes at 37° C. Cells arepelleted, lysed, and a protein assay is performed on the cleared lysate.Equal protein amounts of each sample are subject to SDS-PAGE and westernblotting with either anti-phosphoBtk(Tyr223) antibody (Epitomics, cat.#2207-1) or phosphoBtk(Tyr551) antibody (BD Transduction Labs #558034)to assess Btk autophosphorylation or an anti-Btk antibody (BDTransduction Labs #611116) to control for total amounts of Btk in eachlysate.

Example 903 B-Cell Proliferation Assay

A generalized procedure for a standard cellular B-cell proliferationassay that can be used to test Formula I compounds is as follows.B-cells are purified from spleens of 8-16 week old Balb/c mice using aB-cell isolation kit (Miltenyi Biotech, Cat #130-090-862). Testingcompounds are diluted in 0.25% DMSO and incubated with 2.5×10⁵ purifiedmouse splenic B-cells for 30 min prior to addition of 10 μg/ml of a goatF(ab′)2 anti-mouse IgM antibody (Southern Biotech Cat #1022-14) in afinal volume of 100 μl. Following 52 hr incubation, 1 μCi ³H-thymidineis added and plates are incubated an additional 16 hr prior to harvestusing the manufacturer's protocol for SPA[³H] thymidine uptake assaysystem (Amersham Biosciences # RPNQ 0130). SPA-bead based fluorescenceis counted in a microbeta counter (Wallace Triplex 1450, Perkin Elmer).

Example 904 T Cell Proliferation Assay

A generalized procedure for a standard T cell proliferation assay thatcan be used to test Formula I compounds is as follows. T cells arepurified from spleens of 8-16 week old Balb/c mice using a Pan T cellisolation kit (Miltenyi Biotech, Cat #130-090-861). Testing compoundsare diluted in 0.25% DMSO and incubated with 2.5×10⁵ purified mousesplenic T cells in a final volume of 100 μl in flat clear bottom platesprecoated for 90 min at 37° C. with 10 μg/ml each of anti-CD3 (BD#553057) and anti-CD28 (BD #553294) antibodies. Following 24 hrincubation, 1 μCi ³H-thymidine is added and plates incubated anadditional 36 hr prior to harvest using the manufacturer's protocol forSPA[³H] thymidine uptake assay system (Amersham Biosciences # RPNQ0130). SPA-bead based fluorescence was counted in a microbeta counter(Wallace Triplex 1450, Perkin Elmer).

Example 905 CD86 Inhibition Assay

A generalized procedure for a standard assay for the inhibition of Bcell activity that can be used to test Formula I compounds is asfollows. Total mouse splenocytes are purified from spleens of 8-16 weekold Balb/c mice by red blood cell lysis (BD Pharmingen #555899). Testingcompounds are diluted to 0.5% DMSO and incubated with 1.25×10⁶splenocytes in a final volume of 200 μl in flat clear bottom plates(Falcon 353072) for 60 min at 37° C. Cells are then stimulated with theaddition of 15 μg/ml goat F(ab′)2 anti-mouse IgM (Southern Biotech Cat#1022-14), and incubated for 24 hr at 37° C., 5% CO₂. Following the 24hr incubation, cells are transferred to conical bottom clear 96-wellplates and pelleted by centrifugation at 1200×g×5 min. Cells arepreblocked by CD16/CD32 (BD Pharmingen #553142), followed by triplestaining with CD19-FITC (BD Pharmingen #553785), CD86-PE (BD Pharmingen#553692), and 7AAD (BD Pharmingen #51-68981E). Cells are sorted on a BDFACSCalibur® flow cytometer (BD Biosciences, San Jose, Calif.) and gatedon the CD19⁺/7AAD⁻ population. The levels of CD86 surface expression onthe gated population is measured versus test compound concentration.

Example 906 B-ALL Cell Survival Assay

The following is a procedure for a standard B-ALL (acute lymphoblasticleukemia) cell survival study using an XTT readout to measure the numberof viable cells. This assay can be used to test Formula I compounds fortheir ability to inhibit the survival of B-ALL cells in culture. Onehuman B-cell acute lymphoblastic leukemia line that can be used isSUP-B15, a human Pre-B-cell ALL line that is available from the ATCC.

SUP-B15 pre-B-ALL cells are plated in multiple 96-well microtiter platesin 100 μl of Iscove's media+20% FBS at a concentration of 5×10⁵cells/ml. Test compounds are then added with a final conc. of 0.4% DMSO.Cells are incubated at 37° C. with 5% CO₂ for up to 3 days. After 3 dayscells are split 1:3 into fresh 96-well plates containing the testcompound and allowed to grow up to an additional 3 days. After each 24 hperiod, 50 μl of an XTT solution is added to one of the replicate96-well plates and absorbance readings are taken at 2, 4 and 20 hoursfollowing manufacturer's directions. The reading taken with an OD forDMSO only treated cells within the linear range of the assay (0.5-1.5)is then taken and the percentage of viable cells in the compound treatedwells are measured versus the DMSO only treated cells.

Example 907 CD69 Whole Blood Assay

Human blood is obtained from healthy volunteers, with the followingrestrictions: 1 week drug-free, non-smokers. Blood (approximately 20 mlsto test 8 compounds) is collected by venipuncture into Vacutainer®(Becton, Dickinson and Co.) tubes with sodium heparin.

Solutions of Formula I compounds at 10 mM in DMSO are diluted 1:8 in100% DMSO, then are diluted by three-fold serial dilutions in 100% DMSOfor a ten point dose-response curve. The compounds are further diluted1:12.5 in H₂O and then an aliquot of 5.5 μl of each compound is added induplicate to a 2 ml 96-well square top/tapered V-bottom deep-well plates(Catalog No. 59623-23; Analytical Sales and Services; Pompton Plains,N.J.); 5.5 μl of 8% DMSO in H₂O is added as control and no-stimuluswells. Human whole blood—HWB (100 μl) is added to each well. Aftermixing the plates are incubated at 37° C., 5% CO₂, 100% humidity for 60minutes. Goat F(ab′)₂ anti-human IgM (Southern Biotech Cat#2022-14, 10μl of a 500 μg/ml solution, 50 μg/ml final) is added to each well(except the no-stimulus wells) with mixing and the plates are incubatedfor an additional 16 hours. At the end of the 16 hour incubation,samples are incubated with fluorescent labeled antibodies for 30minutes, at 37° C., 5% CO₂, 100% humidity. Include induced control,unstained and single stains for compensation adjustments and initialvoltage settings. Samples are then lysed with PharM Lyse™ (BDBiosciences Pharmingen) according to the manufacturer's instructions andfixed in 1% paraformaldehyde in PBS containing 1% bovine serum albumine.Samples are then transferred to a 96 well plate suitable to be run onthe BD Biosciences HTS 96 well system on the LSRII machine. Dataacquired and Mean Fluorescence Intensity values were obtained using BDBiosciences DIVA Software. Results are initially analyzed by FACSanalysis software (Flow Jo). The inhibitory concentrations (IC50, IC70,IC90, etc.) for test compounds is defined as the concentration whichdecreases by, for example 50%, the mean fluorescence intensity of CD69expression on cells that are also CD19 positive and CD27 negativestimulated by anti-IgM. The IC70 values are calculated by GenedataScreener, using a nonlinear regression curve fit and are shown in Tables1 and 2.

Example 908 In Vitro Cell Proliferation Assay

Efficacy of Formula I compounds are measured by a cell proliferationassay employing the following protocol (Mendoza et al (2002) Cancer Res.62:5485-5488). The CellTiter-Glo® Luminescent Cell Viability Assay,including reagents and protocol are commercially available (PromegaCorp., Madison, Wis., Technical Bulletin TB288). The assay assesses theability of compounds to enter cells and inhibit cell proliferation. Theassay principle is based on the determination of the number of viablecells present by quantitating the ATP present in a homogenous assaywhere addition of the Cell-Titer Glo reagent results in cell lysis andgeneration of a luminescent signal through the luciferase reaction. Theluminescent signal is proportional to the amount of ATP present.

A panel of B-cell lymphoma cell lines (e.g. BJAB, SUDHL-4, TMD8,OCI-Ly10, OCI-Ly3, WSU-DLCL2) are plated into 384-well plate in normalgrowth medium, and serially diluted BTK inhibitors or DMSO alone wereadded to each well. Cell viability is assessed after 96 hour incubationby CellTiter-Glo® (Promega). Data may be presented as Relative cellviability in BTK inhibitor-treated cells relative to DMSO-treatedcontrol cells. Data points are the mean of 4 replicates at each doselevel. Error bars represent SD from the mean.

Procedure: Day 1—Seed Cell Plates (384-well black, clear bottom,microclear, TC plates with lid from Falcon #353962), Harvest cells, Seedcells at 1000 cells per 54 μl per well into 384 well Cell Plates for 3days assay. Cell Culture Medium: RPMI or DMEM high glucose, 10% FetalBovine Serum, 2 mM L-Glutamine, P/S. Incubate O/N at 37° C., 5% CO2.

Day 2—Add Drug to Cells, Compound Dilution, DMSO Plates (serial 1:2 for9 points), Add 20 μl compounds at 10 mM in the 2nd column of 96 wellplate. Perform serial 1:2 across the plate (10 μl+20 μl 100% DMSO) for atotal of 9 points using Precision. Media Plates 96-well conical bottompolypropylene plates from Nunc (cat.#249946) (1:50 dilution) Add 147 μlof Media into all wells. Transfer 3 μl of DMSO+compound from each wellin the DMSO Plate to each corresponding well on Media Plate usingRapidplate®.

Drug Addition to Cells, Cell Plate (1:10 dilution), Add 6 μl ofmedia+compound directly to cells (54 μl of media on the cells already).Incubate 3 days at 37 C, 5% CO2 in an incubator that will not be openedoften.

Day 5—Develop Plates, Thaw Cell Titer Glo Buffer at room temperature.Remove Cell Plates from 37° C. and equilibrate to room temperature. forabout 30 minutes. Add Cell Titer Glo Buffer to Cell Titer Glo Substrate(bottle to bottle). Add 30 μl Cell Titer Glo Reagent (Promega cat.#G7572) to each well of cells. Place on plate shaker for about 30minutes. Read luminescence on Analyst HT Plate Reader (half second perwell).

Cell viability assays and combination assays: Cells were seeded at1000-2000 cells/well in 384-well plates for 16 h. On day two, nineserial 1:2 compound dilutions are made in DMSO in a 96 well plate. Thecompounds are further diluted into growth media using a Rapidplate®robot (Zymark Corp., Hopkinton, Mass.). The diluted compounds are thenadded to quadruplicate wells in 384-well cell plates and incubated at37° C. and 5% CO2. After 4 days, relative numbers of viable cells aremeasured by luminescence using Cell-Titer Glo (Promega) according to themanufacturer's instructions and read on a Wallac Multilabel Reader®(PerkinElmer, Foster City). EC50 values are calculated using Prism® 4.0software (GraphPad, San Diego). Formula I compounds and chemotherapeuticagents are added simultaneously or separated by 4 hours (one before theother) in all assays.

An additional exemplary in vitro cell proliferation assay includes thefollowing steps:

1. An aliquot of 100 μl of cell culture containing about 10⁴ cells inmedium is deposited in each well of a 384-well, opaque-walled plate.

2. Control wells are prepared containing medium and without cells.

3. The compound is added to the experimental wells and incubated for 3-5days.

4. The plates are equilibrated to room temperature for approximately 30minutes.

5. A volume of CellTiter-Glo Reagent equal to the volume of cell culturemedium present in each well is added.

6. The contents are mixed for 2 minutes on an orbital shaker to inducecell lysis.

7. The plate is incubated at room temperature for 10 minutes tostabilize the luminescence signal.

8. Luminescence is recorded and reported in graphs as RLU=relativeluminescence units.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. Accordingly, all suitablemodifications and equivalents may be considered to fall within the scopeof the invention as defined by the claims that follow. The disclosuresof all patent and scientific literature cited herein are expresslyincorporated in their entirety by reference.

We claim:
 1. A compound selected from Formula I:

or stereoisomers, tautomers, or pharmaceutically acceptable saltsthereof, wherein: X¹ is CR¹ or N; X² is CR² or N; X³ is CR³ or N; R¹, R²and R³ are independently selected from H, F, Cl, CN, —NH₂, —NHCH₃,—N(CH₃)₂, —OH, —OCH₃, —OCH₂CH₃, —OCH₂CH₂OH, and C₁-C₃ alkyl; X⁴, X⁵, X⁶,and X⁷ are independently selected from CH and N; Y¹ and Y² areindependently selected from CH and N; Z is O or NR, where R is H orC₁-C₃ alkyl; Q is selected from the groups having the structure:

where R⁴ is selected from —CH═CH₂, —C(CH₃)═CH₂, —C(CN)═CH₂, —C≡CCH₃, and—C≡CH; and R⁵ is selected from H and C₁-C₃ alkyl; R^(6a), R^(6b),R^(7a), and R^(7b) are independently selected from H, F, Cl, CN, —NH₂,—NHCH₃, —N(CH₃)₂, —OH, —OCH₃, —OCH₂CH₃, —OCH₂CH₂OH, and C₁-C₃ alkyl; orR^(6a) and R^(7a) form a five-, six-, or seven-membered carbocyclyl orheterocyclyl ring; or R⁵ and R^(6a) form a five-, six-, orseven-membered heterocyclyl ring; or if Z is nitrogen, then Z andR^(7a), or Z and R^(6a) form a five-, six-, or seven-memberedheterocyclyl ring; R⁸ is selected from H, F, Cl, CN, —CH₂OH, —CH(CH₃)OH,—C(CH₃)₂OH, —CH(CF₃)OH, —CH₂F, —CHF₂, —CH₂CHF₂, —CF₃, —C(O)NH₂,—C(O)NHCH₃, —C(O)N(CH₃)₂, —NH₂, —NHCH₃, —N(CH₃)₂, —NHC(O)CH₃, —OH,—OCH₃, —OCH₂CH₃, —OCH₂CH₂OH, cyclopropyl, cyclopropylmethyl,1-hydroxycyclopropyl, imidazolyl, pyrazolyl, 3-hydroxy-oxetan-3-yl,oxetan-3-yl, and azetidin-1-yl; R⁹ is selected from the structures:

where the wavy line indicates the site of attachment; and where alkyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl are optionallysubstituted with one or more groups independently selected from F, Cl,Br, I, —CN, —CH₃, —CH₂CH₃, —CH(CH₃)₂, —CH₂CH(CH₃)₂, —CH₂OH, —CH₂OCH₃,—CH₂CH₂OH, —C(CH₃)₂CH, —CH(OH)CH(CH₃)₂, —C(CH₃)₂CH₂OH, —CH₂CH₂SO₂CH₃,—CH₂OP(O)(OH)₂, —CH₂F, —CHF₂, —CF₃, —CH₂CF₃, —CH₂CHF₂, —CH(CH₃)CN,—C(CH₃)₂CN, —CH₂CN, —CO₂H, —COCH₃, —CO₂CH₃, —CO₂C(CH₃)₃, —COCH(OH)CH₃,—CONH₂, —CONHCH₃, —CON(CH₃)₂, —C(CH₃)₂CONH₂, —NH₂, —NHCH₃, —N(CH₃)₂,—NHCOCH₃, —N(CH₃)COCH₃, —NHS(O)₂CH₃, —N(CH₃)C(CH₃)₂CONH₂,—N(CH₃)CH₂CH₂S(O)₂CH₃, —NO₂, ═O, —OH, —OCH₃, —OCH₂CH₃, —OCH₂CH₂OCH₃,—OCH₂CH₂OH, —OCH₂CH₂N(CH₃)₂, —OP(O)(OH)₂, —S(O)₂N(CH₃)₂, —SCH₃,—S(O)₂CH₃, —S(O)₃H, cyclopropyl, oxetanyl, azetidinyl,1-methylazetidin-3-yl)oxy, N-methyl-N-oxetan-3-ylamino,azetidin-1-ylmethyl, pyrrolidin-1-yl, and morpholino.
 2. The compound ofclaim 1 wherein X¹ is N.
 3. The compound of claim 1 wherein X² is N. 4.The compound of claim 1 wherein X³ is N.
 5. The compound of claim 1wherein X¹ and X³ are N, X¹ and X² are N, or X² and X³ are N.
 6. Thecompound of claim 1 wherein X¹ and X³ are CH, and X² is CF.
 7. Thecompound of claim 1 wherein X⁴ is N.
 8. The compound of claim 1 whereinX⁴ and X⁵ are N.
 9. The compound of claim 1 wherein Y¹ is CH and Y² isN.
 10. The compound of claim 1 wherein Y¹ is N and Y² is CH.
 11. Thecompound of claim 1 wherein Y¹ and Y² are each CH.
 12. The compound ofclaim 1 wherein R⁴ is —CH═CH₂.
 13. The compound of claim 1 wherein R⁵ isH or —CH₃.
 14. The compound of claim 1 wherein R^(6a), R^(6b), R^(7a),and R^(7b) are H.
 15. The compound of claim 1 wherein R⁸ is —CH₂OH. 16.The compound of claim 1 wherein R⁹ is selected from:


17. The compound of claim 1 selected from Formula Ia:


18. The compound of claim 17 selected from Formula Ib:


19. The compound of claim 17 wherein the group:

is selected from:


20. The compound of claim 1 selected from Formula Ic:


21. The compound of claim 20 selected from Formula Id:


22. The compound of claim 1 selected from:N-{2-[(6-{[5-(2-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-10-yl}-3-(hydroxymethyl)pyridin-4-yl)-1-methyl-2-oxo-1,2-dihydropyridin-3-yl]amino}pyridin-2-yl)oxy]ethyl}prop-2-enamide;N-(cyanomethyl)-1-(4-{[5-(2-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0²⁶]dodeca-2(6),7-dien-10-yl}-3-(hydroxymethyl)pyridin-4-yl)-1-methyl-2-oxo-1,2-dihydropyridin-3-yl]amino}pyrimidin-2-yl)pyrrolidine-3-carboxamide;N-[2-[[6-[[5-[5-fluoro-2-(hydroxymethyl)-3-(4-oxo-6,7,8,9-tetrahydrobenzothiopheno[2,3-d]pyridazin-3-yl)phenyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]oxy]ethyl]prop-2-enamide;N-[2-[[6-[[5-[2-(6-tert-butyl-8-fluoro-1-oxo-phthalazin-2-yl)-3-(hydroxymethyl)-4-pyridyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]oxy]ethyl]prop-2-enamide;N-[2-[[6-[[5-[5-fluoro-2-(hydroxymethyl)-3-(4-oxo-6,7,8,9-tetrahydrobenzothiopheno[2,3-d]pyridazin-3-yl)phenyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]amino]ethyl]prop-2-enamide;and N-[2-[[6-[[5-[5-fluoro-2-(hydroxymethyl)-3-(4-oxo-6,7,8,9-tetrahydrobenzothiopheno[2,3-d]pyridazin-3-yl)phenyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]oxy]ethyl]but-2-ynamide.23. The compound of claim 1 selected from:N-[(1S)-2-[[6-[[5-[2-(7,7-dimethyl-4-oxo-1,2,6,8-tetrahydrocyclopenta[3,4]pyrrolo[3,5-b]pyrazin-3-yl)-3-(hydroxymethyl)-4-pyridyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]oxy]-1-methyl-ethyl]prop-2-enamide;N-[(1S)-2-[[6-[[5-[5-fluoro-2-(hydroxymethyl)-3-(4-oxo-6,7,8,9-tetrahydrobenzothiopheno[2,3-d]pyridazin-3-yl)phenyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]oxy]-1-methyl-ethyl]prop-2-enamide;N-[2-[[6-[[5-[2-(7,7-dimethyl-4-oxo-1,2,6,8-tetrahydrocyclopenta[3,4]pyrrolo[3,5-b]pyrazin-3-yl)-3-(hydroxymethyl)-4-pyridyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]amino]ethyl]prop-2-enamide;N-[2-[[6-[[5-[2-(7,7-dimethyl-4-oxo-1,2,6,8-tetrahydrocyclopenta[3,4]pyrrolo[3,5-b]pyrazin-3-yl)-3-(hydroxymethyl)-4-pyridyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]-methyl-amino]ethyl]prop-2-enamide;N-[2-[[6-[[5-[5-fluoro-2-(hydroxymethyl)-3-(4-oxo-6,7,8,9-tetrahydrobenzothiopheno[2,3-d]pyridazin-3-yl)phenyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]-methyl-amino]ethyl]prop-2-enamide;N-[2-[[6-[[5-[2-(7,7-dimethyl-4-oxo-1,2,6,8-tetrahydrocyclopenta[3,4]pyrrolo[3,5-b]pyrazin-3-yl)-3-(hydroxymethyl)-4-pyridyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]oxy]ethyl]-N-methyl-prop-2-enamide;N-[2-[[6-[[5-[5-fluoro-2-(hydroxymethyl)-3-(4-oxo-6,7,8,9-tetrahydrobenzothiopheno[2,3-d]pyridazin-3-yl)phenyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]oxy]ethyl]-N-methyl-prop-2-enamide;N-[(3S)-1-[6-[[5-[2-(7,7-dimethyl-4-oxo-1,2,6,8-tetrahydrocyclopenta[3,4]pyrrolo[3,5-b]pyrazin-3-yl)-3-(hydroxymethyl)-4-pyridyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]-3-piperidyl]prop-2-enamide;N-[(3S)-1-[6-[[5-[5-fluoro-2-(hydroxymethyl)-3-(4-oxo-6,7,8,9-tetrahydrobenzothiopheno[2,3-d]pyridazin-3-yl)phenyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]-3-piperidyl]prop-2-enamide;N-[2-[[6-[[5-[2-(7,7-dimethyl-4-oxo-1,2,6,8-tetrahydrocyclopenta[3,4]pyrrolo[3,5-b]pyrazin-3-yl)-3-(hydroxymethyl)-4-pyridyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]oxy]ethyl]but-2-ynamide;2-cyano-N-[2-[[6-[[5-[5-fluoro-2-(hydroxymethyl)-3-(4-oxo-6,7,8,9-tetrahydrobenzothiopheno[2,3-d]pyridazin-3-yl)phenyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]oxy]ethyl]prop-2-enamide;and2-cyano-N-[2-[[6-[[5-[2-(7,7-dimethyl-4-oxo-1,2,6,8-tetrahydrocyclopenta[3,4]pyrrolo[3,5-b]pyrazin-3-yl)-3-(hydroxymethyl)-4-pyridyl]-1-methyl-2-oxo-3-pyridyl]amino]-2-pyridyl]oxy]ethyl]prop-2-enamide.24. A pharmaceutical composition comprised of a compound of claim 1 anda pharmaceutically acceptable carrier, glidant, diluent, or excipient.25. A process for making a pharmaceutical composition which comprisescombining a compound of claim 1 with a pharmaceutically acceptablecarrier, glidant, diluent, or excipient.
 26. A method of treating adisease or disorder which comprises administering a therapeuticallyeffective amount of the pharmaceutical composition of claim 24 to apatient with a disease or disorder selected from systemic and localinflammation, arthritis, inflammation related to immune suppression,organ transplant rejection, allergies, ulcerative colitis, Crohn'sdisease, dermatitis, asthma, systemic lupus erythematosus, extra-renallupus, Sjögren's Syndrome, multiple sclerosis, scleroderma/systemicsclerosis, idiopathic thrombocytopenic purpura (ITP), anti-neutrophilcytoplasmic antibodies (ANCA) vasculitis, chronic obstructive pulmonarydisease (COPD), and psoriasis.
 27. The method of claim 26 wherein thedisease or disorder is rheumatoid arthritis.
 28. The method of claim 26further comprising administering an additional therapeutic agentselected from an anti-inflammatory agent, an immunomodulatory agent,chemotherapeutic agent, an apoptosis-enhancer, a neurotropic factor, anagent for treating cardiovascular disease, an agent for treating liverdisease, an anti-viral agent, an agent for treating blood disorders, anagent for treating diabetes, and an agent for treating immunodeficiencydisorders.
 29. The method of claim 28 wherein the additional therapeuticagent is a Bcl-2 inhibitor.
 30. The method of claim 29 wherein the Bcl-2inhibitor is venetoclax.
 31. The method of claim 28 wherein theadditional therapeutic agent is a Btk inhibitor.
 32. The method of claim31 wherein the Btk inhibitor is ibrutinib.
 33. The method of claim 28wherein the additional therapeutic agent is a JAK inhibitor.
 34. Themethod of claim 28 wherein the additional therapeutic agent is ananti-CD20 antibody.
 35. The method of claim 34 wherein the anti-CD20antibody is obinutuzumab or rituximab.
 36. A kit for treating acondition mediated by Bruton's tyrosine kinase, comprising: a) apharmaceutical composition of claim 24; and b) instructions for use.